; 593 
116 
lopy 1 



The Organic Matter of the Soil: A Study of 

the Nitrogen Distribution in Different 

Soil Types 



A THESIS SUBMITTED TO THE FACULTY OF THE GRADUATE 
SCHOOL OF THE UNIVERSITY OF MINNESOTA 

BY 

CLARENCE AUSTIN MORROW, B.S., M.A. 

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE 
DEGREE OF DOCTOR OF PHILOSOPHY 

JUNE, 1918 



MINNEAPOLIS 
UNIVERSITY PRINTING CO. 



The Organic Matter of the Soil: A Study of 

the Nitrogen Distribution in Different 

Soil Types 



A THESIS SUBMITTED TO THE FACULTY OF THE GRADUATE 
SCHOOL OF THE UNIVERSITY OF MINNESOTA 

BY 

CLARENCE AUSTIN MORROW, B.S, M.A. 

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE 
DEGREE OF DOCTOR OF PHILOSOPHY 

JUNE, 1918 



MINNEAPOLIS 
UNIVERSITY PRINTING CO. 



ACKNOWLEDGMENT. 

This investigation was carried out under the direction of Doc- 
tor Ross Aiken Gortner. The author takes this opportunity to 
express his appreciation and gratitude for the help extended during 
the prosecution of this work, and for the unfailing kindness, 
thoughtful advice and encouragement which was given. 

C. A. M. 

University of Minnesota. 
Division Of Soils. 
January 1917. 



TABLE OF CONTENTS. 

Page 
I. Introduction: Our present knowledge of the organic matter of 

the soil 7 

A. Early investigations 7 

B. The humus theory of Grandeau 12 

C. The complexity of the ammonia soluble material....... 14 

D. The presence of definite organic compounds in the soil.. 15 

E. The origin of organic compounds in the soil 16 

1. Nucleoprotein decomposition 17 

2. Nucleic acid decomposition 17 

3. Lecithin decomposition .' 18 

F. Bacterial processes which influence the form of soil 

nitrogen 19 

1. Deamination or reduction 20_ 

2. Decarboxylation or amine formation 20 

3. Hydrolysis 20 

4. Oxidation 21 

G. Nitrogen distribution in the soil 21 

H. A summary of the nature of the organic matter of the 

soil in the light of our present knowledge 29 

II. Experimental: A study of the nitrogen distribution in dififerent 

soil types 31 

A. The problem 31 

B. The material 31 

1. ■ Calcareous black grass-peat 31 

2. Sphagnum-covered peat 31 

3. Acid "muck" soil , 31 

4. Fargo clay loam 32 

5. Fargo silt loam 32 

6. Carrington silt loam 32 

7. Hempstead silt loam 32 

8. Prairie--covered loess ^2 

9. Forest-covered loess 32 

10. Hempstead silt loam subsoil 32 

C The method ^-^ 

1. The method in detail for a peat soil 34 

2. The method in detail for a mineral soil 36 

3. The method for determination of "Jodidi num- 

bers" ■^' 

4. The determination of nitrogen 38 

4. The analytical data 38 

1. The analysis of "fibrin from blood" hydrolyzed in 

the presence of 100 grams of ignited subsoil 38 

2. Calcareous black grass-peat 41 

3. Sphagnum-covered peat 42 

4. Acid "muck" soil 42 



6 

5. Fargo clay loam 43 

6. Fargo silt loam 44 

7. Carrington silt loam 45 

8. Hempstead silt loam 45 

9. Prairie-covered loess 46 

10. Forest-covered loess 47 

11. Sphagnum-covered peat hydrolyzed in the pres- 

ence of nine times its weight of a mineral 
subsoil 47 

12. Sphagnum-covered peat hydrolyzed in the pres- 

ence of metallic tin 49 

13. Analysis of a 1 per cent, hydrochloric acid extract 

of sphagnum-covered peat and (in part) of 
calcareous black grass-peat 50 

14. Analysis of a portion of sphagnum-covered peat 

soluble in 4 per cent, sodium hydroxide and 
precipitated by hydrochloric acid and (in 
part) of a similar solution from a calcareous 
black grass-peat 52 

15. Analysis of a portion of sphagnum-covered peat 

soluble in 4 per cent sodium hydroxide and 
not precipitated by hydrochloric acid and (in 
part) of a similar solution from a calcareous 
black grass-peat 54 

16. "Jodidi numbers" 55 

17. Summary tables 57 

18. An attempt to isolate pure proteins from a soil 59 

a. Extraction with 70 per cent ethyl alcohol 59 

b. Extraction with absolute alcohol 60 

c. Extraction with 10 per cent sodium chlor- 

ide 61 

III. Discussion 62 

A. Changes in nitrogen distribution in a protein when hy- 
drolyzed in the. presence of a mineral soil 62 

B. The human nitrogen, its origin, and significance. ........ 63 

C. The effect of the quantity of acid used for the hydrolysis 
on the amount of nitrogen dissolved and the nitrogen 
distribution in soils 66 

D. The percentage of soil nitrogen extracted by acid hy- 
drolysis 66 

E. "Jodidi numbers" 67 

F. Attempts to extract proteins from the soil 67 

G. A consideration of the nitrogen distribution in different 
extracts from the sphagnum-covered peat 67 

H. General conclusions in regard to the distribution of soil 

nitrogen in different soil types 68 

IV. Summary 70 

V. Literature cited 72 

Biographical 80 



I. INTRODUCTION: OUR PRESENT KNOWLEDGE OF 
THE ORGANIC MATTER OF THE SOIL. 

A. Early Investigations. 

It has been recognized from the time of tlie alchemists that 
manures are of fundamental importance for the growth of plants. 
The alchemists believed that by a process of transmutation water 
was converted into plant tissue. In an attempt to prove this an 
interesting experiment was conducted by van Helmont (1648). 
In a large earthen vessel he placed 200 pounds of dry earth, and 
planted in it a small willow tree weig'hing five pounds, and for five 
years he watered the plant either with rain or distilled water. At 
the end of that time he pulled up the willow and found that it 
weighed 169 pounds and three ounces. The dry soil remaining after 
the experiment was found to have lost only two ounces. He drew 
the apparently justifiable conclusion that 164 pounds of i^oots, bark, 
leaves, and branches had been produced by direct transmutation 
of water. 

It is evident that it was essential to establish the composition 
of water and some of the components of the air before further 
work could have real value. 

Not until the discovery of oxygen by Scheele (1777) and 
the proof of the composition of water by Cavendish (1784), as well 
as the work of de Saussure (1804) regarding- the role played by 
carbon dioxide in plant and animal life, did we have any real 
knowledge concerning the sources of matter stored up in plants. 

During- the first quarter of the nineteenth century organic com- 
pounds were regarded as capable of being S3aithesized only in the 
living cells of plants or animals. This idea that organic compounds 
could be formed only through a special vital force was overthrown 
by the classic work of Wohler (1828) when he prepared urea, a 
purely animal product*, by evaporating- ammonium cyanate to 
dryness. This fact attracted the attention of chemists, and prac- 
tically all work done from that time until 1840 was on some phase 
of organic chemistry. This overthrow of the belief in a vital force 
and the improved method of organic analysis by Berzelius (1808- 
18) paved the way for a more thorough understanding of the part 
taken by the organic matter in the soil, and consequently created 
a renewed interest in scientific investigations relating to agricul- 
ture. 

Some important work had been accomplished previous to this 
time. The most valuable was that of de Saussure's (1804) "Re- 
cherches chimiques sur la vegetation" which was the first syste- 
matic work showing the source of compounds stored up in the 
plant. He pointed out that the quantitative increase in the carbon, 
hydrogen, and oxygen, when plants were exposed to sunlight, was 

*Fosse (1912) notes the presence of lu-ea in certain plants. 



due to the cari)on dioxide of the air and water of the soil. He be- 
lieved that the nitrogen of the soil was the chief source of the 
nitrogen found in plants. Unfortunately his conclusions were 
not accepted at that time, and it was not until about fifty years 
later, when other investigators had repeated his experiments, that 
his results were finally accepted by botanists and chemists. 

One of the first investigators to see the relation between chem- 
istry and agriculture was that of Sir Humphry Davy (1813), who 
published a book entitled, "Elements of Agricultural Chemistry." 
This treated of the composition of air, soil, manures, plants, and 
of the influence of heat and light upon the growth of plants. 

Thaer ( 1809-10) contended that humus determined the fertility 
of the soil, that plants obtained their food mainly from humus, 
and that the carbon compounds of plants were produced from the 
organic carbon compounds of the soil. These ideas gave rise to his 
so-called humus theory, which was later shown to be inadequate. 
His writings, however, did much to stimulate later investigation. 

The French investigator Boussingault verified much of the 
earlier work of de Saussure and secured many additional facta 
concerning the chemistry of growth. His predecessors had sought 
to solve the cjuestion as to whether plants assimilate the free or 
uncombined nitrogen of the atmosphere. Boussingault (1838, 
1838a) improved the methods for the determination of the point in 
question, and showed that peas and clover could get their nitrogen 
from the air while wheat could not. Unfortunately, he did not 
make as much of this discovery as he might have done. 

Boussingault was the first to^ have a chemical laboratory lo- 
cated on a farm and to make investigations along a practical line 
in connection with agriculture. His was the first agricultural ex- 
periment station. 

The investigations of de Saussure, Boussingault, Davy, Thaer, 
and others paved the way for the work and writings of Liebig. He 
published (1840) "Organic Chemistry in its Application to Agri- 
culture and Physiology," which was an important factor in at- 
tracting the attention of the public to agricultural problems. Many 
.of his investigations and discoveries in the field of organic chem- 
istry were applied directly to his interpretation of these problems. 

He assailed the humus theory of Thaer and showed that humus 
could not be an adequate source of the plant's carbon. By applying 
the exact methods of chemistry to agriculture Liebig succeeded in 
establishing that plants derive the carbon of their tissues from 
the carbon" dioxide of the air, and not from the carbon compounds 
that may be present in the soil. He came to regard the ammonia 
of the air as analogous with the carbon .dioxide of the air, and 
preached the doctrine that plants were able to derive their nitrogen- 
ous food from the atmosphere. In the Farmer's Magazine, for in- 
stance, he writes : 

If the soil be suitable, if it contains a sufficient quantity of alkalis, phos- 
phates, and sulphates, nothing will be wanting. The plants will derive their 
ammonia from the atmosphere as they do carbonic acid. (Cited by Russell, 
1912.) 

Although the work of Liebig was not conducted in connection 
with field experiments, it had a stimulating effect upon agricultural 



investig-ation, and we are greatly indebted to him for summarizing 
previous work and pointing out valuable lines of future research. 
In his book (1840) he states, that — 

a rational system of agriculture cannot be formed witliout the application 
of scientific principles, for such a system must be based on an exact ac- 
Quaintance with the means of nutrition of vegetables, and with the influence 
of soils, and actions of manures upon them. This knowledge we must seek 
from chemistry, which teaches the mode of investigating- the composition and 
study of the character of the different substances from which plants derive 
their nourishment. 

In one essential point, however, he fell into error. Lawes, the 
pioneer experimenter on agriculture in England, flatly denied the 
accuracy of Liebig's conclusions as regards nitrogen assimilation. 
The results of the investigations at Rothamsted as conducted by 
Lawes and Gilbert (1851) on the non-assimilation of atmospheric 
nitrogen by crops, were accepted as conclusive evidence upon this 
much discussed question. 

The alkali soluble portion of the organic matter of the soil 
has formed for many years the subject of keen interest and discus- 
sion. This portion of the soil organic matter was called "humus" 
by the earlier writers, but this name has in more recent times been 
used by some American and most European investigators to desig- 
nate the total organic matter of the soil. I have used the term 
throughout this paper in its original meaning. In early days the 
"humus" was regarded as being of very simple composition. De 
Saussure (1804) for instance, described it as a "brown combustible 
powder soluble' in alkalies and ammonia compounds." 

Klaproth* applied the name "ulmin" to dark colored amorphous 
bodies such as those obtained by Vauquelin (1797) from the bark 
of diseased elm trees. Sprengel (De Candolle** 1833, p. 280), who 
obtained similar bodies from soils applied to these the name "humic 
acid." Berzelius (1838) evidently had the general meaning of the 
term "humus" in mind when he used the term "humin" in describ- 
ing certain dark colored constituents of vegetable mold. Follow- 
ing the use of the term "humin" as applied to what was considered 
to be a definite organic body, a number of other workers took up 
the study of similar substances, and a number of other terms more 
or less related, soon appeared. The name of Mulder (1849) is asso- 
ciated for the most part with the terms applied to humus-like sub- 
stances which have appeared more or less in the literature from that 
time to the present. For instance, he says : 

At present seven different organic substances are known to exist in the 
soil. They are crenic acid, apocrenic acid, geic acid, humic acid, and humin, 
ulmic acid and ulmin. 

These bodies were divided by him into two groups, one con- 
sisting of crenic and apocrenic acids, and the other group embrac- 
ing all the others. According to Mulder (1849) these seven or- 
ganic bodies were intimately related, and five at least were five suc- 
cessive steps in the decay of organic matter in the soil. The first 
step in this decay he regarded as ulmic acid ; this on further 
oxidation yielded humic acid ; and this in its turn, on still further 
oxidation, geic acid. Continued oxidation produced apocrenic, 
and finally crenic acid. 

*(De Candolle 1833, p. 279) states "Das Ulmin ist von Klaproth entdeckt 
worben." 

** I have been unable to verify the original citation, which, according to 
Proper was Kastner's Archiv. Bd. 7, p. 163; Bd. 8, p. 145. Presumably one of 
these articles is that referred to by Russell (1912) entitled "Ueber Pflanzen- 
Naturlehre, Niirnberg, 1826. 



10 

A number of chemists have given the percentage composition 
of the supposed acids, but no two agree. Nothing is knoAvn in re- 
gard to their constitution. The lack of definite chemical character- 
ization of these compounds is stated by Cameron and Bell (1905) 
as follows : 

The existence itself of these acids has never been satisfactorily demon- 
strated. * * * * No satisfactory description of the physical or chemical 
properties of these supposed acids, their salts, or characteristic derivatives, 
have been recorded. 

Nearly every writer on soils from the time of Mulder to Hil- 
gard (1906, p. 126) spoke of these acids with the same assurance 
as of oxalic or tartaric acids, or any other organic compound that 
has well known derivatives. 

The early investigators, including Mulder, soon found that 
sugar, starch, carbohydrates generally, and even proteins, when 
treated with strong acids or alkalies, gave rise to dark colored 
compounds having the same general appearance and properties 
as the humus substances arising in the soil through decay. How- 
ever, a conspicuous feature of the work on these humus substances 
is the discordant results for preparations bearing the same name 
and often from the same source. 

Robertson, Irvine, and Dobson (1907) reached conclusions that 
although there were many strong resemblances between natural 
and artificial humus preparations, in regard to properties and com- 
position, yet there are important differences in constitution. Re- 
cently Gortner (1916 c) has shown that in all probability the humin 
formed from carbohydrates is actually formed by a polymerization 
of furfural which is in turn formed from the carbohydrates by the 
action of the acid. Gortner and Blish (1915). Gortner (1916 c), and 
Gortner and Holm (1917) have likewise shown that the dark col- 
ored products originating in an acid hydrolysis of protein sub- 
stances have their origin in the tryptophane nucleus. Obviously, 
if carbohydrate humin originates from furfural, and protein humins 
originate in the tryptophane nucleus, mixtures of protein and 
carbohydrate would produce a great variety of physically similar 
but chemically different mixtures. 

One of the important points at issue between the early in- 
vestigators was whether humic acid and allied bodies contained 
nitrogen as a constituent. Mulder (1849, 1862) held that nitrogen 
was not a constituent of these substances, but was present as 
ammonia, that is the acids were present in the soil as ammonium 
salts, and in this connection he says : 

In good arable soil — that is, one in which the organic constituents are as 
far as possible decomposed — none of these substances contain nitrogen 
as a constituent element: all their nitrogen exists in the state of ammonia. 

Detmer (1871) came to the opposite conclusion, claiming that 
nitrogen in humic acid as usually obtained, was present in organic 
combination (not, however, bound in the humic acid molecule). 
He obtained his humus by digesting with alkali. After precipitat- 
ing Mnth acid, he redissolved it in ammonia and precipitated the 
mineral constituents with phosphoric and oxalic acids and am- 
monium sulphide. After treating with potassium hydroxide and 
precipitating with hydrochloric acid, he obtained a preparation 



11 

containing 1.5 per cent nitrogen. No ammonia was evolved on 
making alkaline, but about 23 per cent of the total nitrogen was 
evolved on treatment with sodium hypobromite. Detnier believed, 
however, that humic acid did not contain nitrogen, but that the 
nitrogen found was present in some organic compound occurring as 
an impurity in his humic acid preparation. By a tedious process 
he was able to lower the nitrogen content of his humic acid to 
0.179 per cent. 

Ritthausen (1877) attributed the high nitrogen content of peat 
to the formation of complex, difficultly decomposable materials by 
absorption of ammonia and pointed to the low ammonia content as 
an indication of it, claiming it was not present as such after absorp- 
tion. In an attempt to disprove Ritthausen's theory. Si vers (1880) 
found that he could expel only very small amounts of ammonia by 
heating with potassium hydroxide. He concluded that all the am- 
monia taken in remained as such, and did not go to form complex 
compounds. He maintained that most of the nitrogen was in the 
form of protein but presented no conclusive evidence. Grouven 
(1883) tried to show that the nitrogen of humus was due to the 
absorption of ammonia by humic acids, but found from various 
samples that only one-fiftieth of the total nitrogen was liberated 
on heating with milk of lime, and only one-twentieth when heated 
for two hours with potassium hydroxide. 

It was found by Loges (1886) that the hydrochloric acid ex- 
tract of the soil gave a precipitate with phosphotungstic acid, which 
is recognized as being a precipitating agent for certain nitrogenous 
compounds. Baumann (1887) found that certain black Russian 
soils rich in humus, containing but small traces of ammonia in the 
soil, gave a considerable amount of it on boiling the soil with dilute 
hydrochloric acid. From this he suggested the presence of amino 
and amide compounds in the soil. About the same time this sub- 
ject was more thoroughly investigated by Berthelot and Andre 
(1886). They found that the nitrogenous matter Avas split up pro- 
ducing ammonia and soluble nitrogenous compounds, and that the 
hydrolysis goes further the greater the strength of the acid, the 
longer it is in contact with the soil and the higher the temperature. 
A soil containing 0.174 per cent of nitrogen was heated on a water 
bath for two hours with 7 per cent hydrochloric acid and 31.9 per 
cent of its nitrogen was dissolved. Of this soluble nitrogen 17.8 
per cent was ammonia. Similar experiments were conducted using 
3.4 per cent hydrochloric acid, and 0.7 per cent hydrochloric acid 
and distilled water. 

Warington (1887) working with a sample of Rothamsted soil, 
Avhich had been heavily manured, showed the presence of a small 
amount of amide nitrogen by using both hypobromite and nitrous 
acid. It seems highly probable from these experiments that at 
least a part of the nitrogen in the soil is present as amino com- 
pounds. Eggertz (1888) found that the nitrogen content of thir- 
teen samples of humus varied from 2.59 to 6.43 per cent and states 
that the nitrogen was present in organic form and not as an am- 
monium salt. Sestini (1899) also showed the presence of amino 



12 

nitrogen by the action of nitrous acid. Dojarenko (1902) working 
with humus from seven Russian soils found appreciable quantities 
of amino nitrogen. Unlike previous investigators he determined 
the amount present quantitatively, and assumed all of the 
amino nitrogen was present as amino acids. He also made deter- 
minations of the ammonia by distillation with magnesium oxide, 
and obtained the amide nitrogen by hydrolysis with dilute hydro- 
chloric acid and subsequent distillation of the ammonia formed 
with magnesium oxide. 

B. The Humus Theory of Grandeau. 

A tremendous impetus was given to. the study of soils by the 
work of Grandeau, because he believed that the ammonia extract 
of soils contained the nutritive substances essential for the life 
of the plant and for the fertility of the soil. The theory that the 
humus extract was of such value had a great deal to do with re- 
tarding the development of the study of the organic matter of the 
soil. 

The method of Grandeau (1872) is essentially the one in use 
at the present time in America for the determination of humus. He 
elaborated a method for the estimation of the "matiere noire" of 
the soil by first leaching the soil with dilute acid in order 
to set the humus free from its combination with the alkaline earths, 
removing the excess of acid by washing with water, then moisten- 
ing the soil with ammonia and allowing it to stand for a short 
time (three to four hours, cf. Grandeau 1877, p. 149), after which 
the humus solution was displaced by repeated washings with am- 
moniacal water. The dark brown solution so obtained was evapor- 
ated to dryness in platinum, weighed, ashed, and the amount of 
"matiere noire" and of ash recorded. Grandeau regarded the humus 
ash as an integral part of the humus. He believed that the organic 
matter which dissolved was responsible for the fertility of the soil, 
apparently not so much because of the carbon and nitrogen con- 
tent, as for the high percentage of phosphoric acid and potash in 
the humus ash. 

The views of Grandeau were never generally adopted in Eu- 
rope although often accepted by individual workers, but have been 
more generally accredited in America, due to the sponsorship of 
certain parts of Grandeau's humus theory by the late Professor 
Hilgard. The American investigators, e. g., Hilgard (1906), Ladd 
(1898), and Snyder (1895, 1897, "and 1901), however, do not report 
the humus ash as an integral part of the humus but call only the 
volatile portion humus. They consider that the humus is, in part 
at least, combined in the soil with inorganic substances ; these 
compounds are called "humates" and to their abundance and pro- 
duction has been ascribed an important part of the maintenance 
of soil fertility. 

Hilgard (1906) regarded the humus of the soil as a definite soil 
product, formed from vegetable material in the soil under the in- 
fluence of fungus and bacterial growths ; this conversion being 



13 

most efficiently carried out in the presence of only a moderate 
amount of moisture, under the influence of a more or less rapid 
circulation of air and in the presence of calcium carbonate to 
neutralize any acids which may be formed. Under these conditions 
the vegetable substance is converted into black, neutral, insoluble 
humus compounds. 

He believed that the nitrogen of plant debris which has become 
an integral part of the soil must first be converted by humifying 
bacteria and fungi into humus before the nitrogen can become avail- 
able to the nitrifying bacteria and thus rendered available for the 
use of the higher plants. His views of the persistence of plant ma- 
terials in soils are contained in the following statement : 

As a matter of course, the several organic compounds contained in plants 
may continue to exist in soils for some time, varying according to conditions 
uf temperature and moisture. Tlius dextrin, glucose, and even lecithin and 
nuclein have been reported to be found. The activity of the numerous fungus 
and bacterial ferments under favoring conditions, will of course, limit the 
continued existence of such compounds somew^iat narrowly so that they can 
hardly be considered as active soil ingredients save in so far as they favor 
the development of bacterial flora. 

Suzuki (1906-08 a) made a study of the formation of humus by 
treating oak leaves with a humus soil and various inorganic com- 
pounds and concluded that not only calcium carbonate but also 
magnesium carbonate promoted the decomposition of moist oak 
leaves by fungi, judging from the amounts of carbon dioxide 
evolved. He further states that the opinion of Hilgard corresponds 
closely to the natural conditions of humification. Further studies 
of Suzuki (1906-08 b) indicate that protein, starch, and pentosans 
contribute to the formation of the black matter of humus, but 
neither fat nor cellulose, and that protection from air is essential. 

One of the latest additions to the idea of specific humification 
of plant materials in the soil is that of Trusov (1915). He inoculat- 
ed various types of organic compounds with soil bacteria for vari- 
ous lengths of time and concludes that humus has its origin in 
lignin, albumen, starch, chlorophyll, and tannic substances; while 
cellulose, hemicelluloses, mono- and disaccharides, glucosides, or- 
g"anic acids including amino acids, and wax forming substances 
do not appear to have any part in its formation. He also finds that 
the org-anic nitrogenous compounds used as nutrients for the micro- 
organisms may serve as an indirect source of humus. 

Weir (1915) has recently questioned the idea that the soluble 
humus of the soil is an indication of the fertility of that soil and 
that the humus nitrogen plays an important role in the nutrition 
of plants. He removed 40 per cent of the nitrogen of the soil by 
extracting the humus with sodium hydroxide and then used the 
extracted soil for pot experiments. However, Gortner (1916 b) 
has shown that in all probability a very considerable portion of the 
humus nitrogen still remained in Weir's extracted soil, for he was 
able to extract 90.3 per cent of the original nitrogen content of 
the soil. This would indicate that nearly all of the soil nitrogen 
could be extracted with sodium hydroxide. 

Snyder (1897) prepared artificial humus by mixing a subsoil 
with certain organic substances and allowing these to remain in a 
moist condition for one year. At the end of the year humus was 



14 

determined on the resulting- mixture by extraction with ammonia, 
following- a previous leaching with dilute acid, and the humus so 
found was regarded as having been formed in the soil during the 
preceding year. Unfortunately Snyder did not correct for am- 
monia soluble org-anic matter in the mixtiwes at the beginning of 
the experiment. The results of Fraps and Hamner (1910) and 
Gortner (1917) show that ammonia dissolves a considerable por- 
tion of material from unchanged organic compounds, so that the 
humus gain at the end of the experiment was in all probability 
actually a loss when compared with the amount of ammonia soluble 
materials at the beginning of the experiment. Fraps and Hamner 
(1910) as well as Gortner (1917) report a series of experiments af- 
ter the general ])lan adopted by Snyder, with the exception that the 
ammonia soluble materials were determined both at the beginning 
and at the end of the experiments, and in each instance the am- 
monia soluble material was found to decrease. 

The experiments of Gortner (1917) furnish no evidence that a 
specific "humiftcation" of plant materials takes place in the soil 
giving rise to an increased amount of "humus." He says: 

On the contrary, all of the evidence is directly opposed to such a con- 
clusion, and it appears altogether probable that the. maximum amount of am- 
monia soluble material is present in a soil immediately after a green manuring 
crop has been plowed under and before the 'humifying' bacteria or fungi 
l>eg'in their work. 

Fraps and Hamner (1910) showed that the humus extract of 
soil must contain substances from unchanged vegetable materials, 
while Gortner (1916 a) pointed out that the extract must contain 
substances from unchanged plant material, from bacteria and 
protozoa. 

C. The Complexity of the Ammonia Soluble Material. 

AVith the development of chemistry the idea has been gradu- 
ally abandoned, that the ammonia soluble compounds can contain 
the whole of the organic matter which is responsible for the fer- 
tility of the' soil. The work of the U. S. Bureau of Soils in the 
isolation of a large number of definite organic compounds from 
the soil, has been a distinct contribution along this line. 

As has been suggested by Gortner (1916 a) — 

If one speculates on the nature of the soil organic matter, it becomes obvi- 
ous that the variety of compounds which are present in a soil is limited 
only by those compounds which were present in the plants growing upon 
the soil, pliis those compounds Avhich compose the bodies of bacteria and 
protozoa, plus the compounds contained in the soil fungi, plus all the various 
compounds which may be formed from the above sources by decay, oxidation, 
and all the intricate chemical reactions which take place in converting dead 
organic material, either into living protoplasm on the one hand, or into 
water, carbon dioxide, and nitrogen on the other. Undoubtedly these organic 
compounds are not the product of 'humification' but are derived from un- 
changed plant material, from protozoa, or from bacteria. 

A part, or all, of these compounds would be found in the 
"humus" extracted l^y Grandeau's method — 

Inasmuch as the 'humus' extract of soils is undoubtedly a mixture of organic 
compounds, many of which are colorless and in all probability are extracted 
from unchanged plant or animal materials, and inasmuch as the soil pigment 
present in this solution probably rarely exceeds 40 per cent of the 'humus a 
determination of 'humus', as ordinarily carried out, appears to be wholly 
without scientific .iustification. (Gortner 1916 a.) 



IS 

D, The Presence of Definite Organic Compounds in the Soil. 

The following types of pure organic compounds have been 
isolated from the soil. In view of Gortner's statement above it is 
of interest to note that all of these compounds are colorless, and 
that nearly all of them were isolated from an alkaline humus ex- 
tract. 

1. Paraffin hydrocarbons. 

Hcntriacontane, CaiH,H. Sclireiner and Shorev (1910 a, 1911 a). 

2. Alcohols 

Argosterol*. QeH440. Schreiner and Shorey (1909, 1909 a) 
Phytosterol, G6H44O, H2O. Schreiner and Shorey (1910 a, 1911 b) 

3. Esters 

"Glycerides of fatty acids." Schreiner and Shorey (1910 a) 
"Resin esters." Schreiner and Shorey (1910 a) 

4. Acids 

Oxalic acid. COOH-COOH. Shorey (1913) 
Succinic acid, COOH-CH2-CH.-COOH. Shorey (1913) 
Saccharic acid, COOH-(CHOH)4-COOH. Shorey (1913) 
Acrylic acid, CH2=CH-COOH. Shorey (1913) 

a-Crotonic acid, CH:,-CH = CH-COOH. Walters and Wise (1916). 

a (l:ll)-Monohvdroxvstearic acid, CH..,(CH:.)XH0H (CH.).,C00]-1. 

Schreiner and Shorey (1910 a, 1910 b) 
Dihydroxystearic acid. CH,(CH2)tCHOH-CHOH(CH.)tCOOH. 

Schreiner and Shorey (1908 a, 1909 a) 
Benzoic acid, GH.COOH. Shorey (1914) 
Metaoxytoluic acid, OH-CsH^-CHs-COOH. Shorey (1914) 
Agroceric acid, C21H42O3. Schreiner and Shorey (1909 a) 
Parafifinic acid, C24H48O2. Schreiner and Shorey (1910 a) 
Lignoccric acid, C24H4s02. Schreiner and Shorey (1910 a) 
"Resin acids" (?). Schreiner and Shorey (1910 a) 

5. Aldehydes 

Salicylic aldehyde, aH4-OH-CHO. Shorey (1913) 

Vanillin, OH-C6H3(OCH3)-CHO. m-methoxy-o-hydroxy benzal- 

dehyde. Shorey (1914) 
Trithiobenzaldehyde, (CeHsCSH)^. Shorey (1913) 

6. Carbohydrates 

Mannitol, CH20H-(CPIOH)4-CH20H. Shorey (1913) 
Rhamnose, CH.-,-(CHOH)4-CHO, H2O. Shorey (1913) 
Pentosan**, GH8O4. Schreiner and Shorey (1910 a), Shorey and 
Lathrop (1910) 

7. Pyrimidine derivatives 

Cytosine. C4H5N3O. 2-oxv. 6-aiTiino pyrimidine. Schreiner and 
Shorey (1910 a, 1910 cj 

8. Purine bases 

Xanthine. C.5H4N4O2. 2, 6-dioxy purine. Schreiner and Shorey 

(1910 a. 1910 c) 
Hypoxanthine, C5H4N4O. 6-oxy purine. Schreiner and Shorey 

(1910 a, 1910 c) 
Adenine. Cr,H,^N5. 6-amino purine. Shorey (1913) 
Guanine***, GH5N5O. 2-amino, 6-oxypurine. Lathrop (1912). 

Schreiner and Lathrop (1912). 
Tetracarbonimid****, C4H4N4O4. Shorey and Walters (1914) 

9. Pyridine derivatives 

" a-Picoline. ^-t-arhoxvlic acid, GFItNO^. Shorey (1906), Schreiner 
and Shorey (1908 b) 

*The composition of the compound was determined by a single analysis, 
made with 0.1500 gram of the substance. It seems highly improbable that 
the composition could be obtained accurately from this amount of material. 

**Pentose sugars have been separated as hydrolysis products of substances 
isolated from the soil (Schreiner and Shorey 1910 a). 

***This compound was isolated from a steam heated soil. 

***» This has been shown to be cyanuric acid (Wise and Walters 1917). 



16 

10. Amines 

Trimethylamine, (CH3)3N. Shorey (1913) 

Choline, HON(CH3)3-CH2-CH20H. Trimethyl oxyethyi am- 
monium hydroxide. Shorey (1913) 

Creatinine, C4H7N3O. fi-im'mo (n) methyl a-keto tetrahydro gly- 
oxalin. Shorey (1911), Schreiner, Shorey, Sullivan and Skinner 
. (1911) 

11. Organic phosphorus compounds 

Nucleic acid. Shorey (1911 a, 1912, and 1913) 
Lecithin. Aso (1904), Stoklasa (1911). 

12. Amino acids 

Arginine, CHi4N402. a-amino, 8-guanidine valerianic acid. 

Schreiner and Shorey (1910 a, 1910 d) 
Histidine. C,iH(,N.,02. a-amino, /^-imidazole propionic acid. 

Schreiner and Shorey (1910 a, 1910 d) 
Lysine, CeHijNiiO-. a £-di-amino, caproic acid. Shorey (1913) 

Whether all of these compounds have actually been isolated 
is perhaps an open question, in view of the minute quantities v^hich 
were obtained, insufficient in many cases for an exact chemical an- 
alysis as stated by Schreiner and Lathrop (1911) : 

The amount of a substance obtained may be so smaU that extreme puri- 
fication is out of the question, and therefore; in such cases, where distinct 
crystaUine form or characteristic tests are not available the identification 
becomes uncertain, as neither melting- point nor analysis can be made. 

E. The Origin of Organic Compounds in the Soil. 

Chardet (1914) gives a discussion of the possible origin of 
certain organic nitrogenous compounds that have been isolated 
from the soil or might be expected to exist. 

A very complete summary of our present knowledge of the or- 
ganic matter of the soil is presented by Jodidi (1914) under these 
headings: I. Introduction; II. The sulphur compounds of the 
soil; III. The influence of certain factors on the quantity of nitro- 
gen contained in the soil; IV. The nature of humus substances ac- 
cording to the older authors ; V. The observations of later authors 
concerning the nature and behavior of humus to certain reagents ; 
VI. Genetic relationship betAveen the chemical compounds in the 
soil and those in plants and animals; VII. The nature of nitrogen 
compounds' in the soil ; VIII. The organic nitrogenous compounds 
of the soil ; IX. Separation of the nitrogenous compounds in a 
sulfuric acid extract (i. e., hydrolysate) of the soil; X. Cleavage 
products of nucleoproteins ; XI. Lecithin products in the soil ; XII. 
Pyridine derivatives in the soil; XIII. Ammonification of amino 
acids and acid amides in the soil ; XIV. The occurrence of hydro- 
carbons, alcohols, and aldehydes in the soil ; and XV. The organic 
acids occurring in the soil. 

Different investigators have succeeded in isolating from soils 
the following nitrogenous compounds which may be related to 
or derived from the proteins : tetracarbonimid, a-picoline y-car- 
boxylic ~acid, trimethylamine, nucleic acid, arginine, histidine, 
lysine, proteoses, and peptones. Potter and Snyder (1915 b) have 
shown that in some soils, at least, free amino acids and peptides 
occur but the amounts are very small. 

Since there have been so many nitrog^enous compounds isolat- 



17 

ed from the soil that are related to proteins, it is well to discuss 
some of the complex organic compounds such as nucleoproteins, 
nucleic acids, and lecithins that find their way to the soil through 
plant and animal remains. While the final decomposition products 
are undoubtedly simple compounds or elements such as carbon 
dioxide, methane, ammonia, nitrogen, and hydrogen, these products 
are reached by fairly definite and well defined methods of cleav- 
age. The process may be a rapid one, a slow one, or one entirely 
arrested at certain stages, all depending on the factors present 
in the soil. 

1. Nucleoprotein decomposition. The nucleoproteins are with- 
out doubt the most complex compounds that enter the soil. They 
are common constituents of plants, animals, bacteria, and molds, and 
hence occur wherever these live or die. The chemical changes 
through which these compounds go during decomposition may be 
rendered clear by the following scheme presented by Lilienfeld 
(1892): 

Nucleoprotein 



Protein Nuclein 

(Histone) | 



Protein Nucleic acid 



The products are then protein and nucleic acid, the latter of 
which has been isolated from the soil (Shorey 1911 a, 1912, and 
1913). 

2. Nucleic acid decomposition. Nucleic acids are constituents 
of all nuclei and on decomposition yield a variety of compounds 
composed of carbon, hydrogen, oxygen, nitrogen, and phosphorus. 
The acids occur in both plant and animal cells. 

Jones (1914) states that all plant nucleic acids contain a pen- 
tose group, while on the other hand all animal nucleic acids yield 
levulinic acid, which is formed from a hexose group in their mole- 
cule. 

The hydrolysis products may be classified, according to Forbes 
and Keith (1914) as follows: 

Nucleic acids 



Pho.sphoric acid Carbohydrates 



Pentoses I 

Hexoses | 

Unidentified Purine Pyrimidine 



Guanine Cytosine 

Adenine Thymine 

Xanthine Uracil 
Hypoxanthine 



18 

The purine and also the pyrimidine derivatives can be changed 
one into the other by chemical means. In a parallel manner much 
the same results can be obtained through the biochemical changes 
brought about by bacteria and enzymes. By chemical agents Kos- 
sel and Steudel ( 1903) transformed cytosine into uracil. In like 
manner Fischer ( 1882) changed guanine into xanthine, and Kossel 
( 1886) changed adenine into hypoxanthine. It was shown by Schit- 
tenhelm and Schroter (1904) that putrefactive bacteria, especially 
those of the coli group, were able to convert guanine and adenine 
into xanthine and hypoxanthine and that bacteria also have the 
ability of breaking down nucleic acid itself. This change of nucleic 
acid is also accomplished by certain enzvmes, the nucleases (of. 
Jones 1914 and Euler 1912). 

It will be clear from the above that the decomposition of 
nucleic acid may take place in many steps and that the intermediate 
as well as the final products may be transformed one into the other. 
This may be accomplished in the soil through the agency of micro- 
organisms or enzymes. Schreiner and Lathrop (1912) working 
with steam heated soils found that from the heated samples less 
nucleic acid was obtained than from the unheated samples. On the 
other hand the decomposition products of nucleic acid were present 
in larger amounts in the heated soil, indicating that hydrolysis of 
nucleic acid has been accomplished in this manner. That nucleic 
acid decomposition does take place in the soil is evidenced by 
the isolation of certain of its decomposition products, e. g., cytosine, 
xanthine, hypoxanthine (Schreiner and Shorey 1910 a, 1910 c), 
adenine (Shorey 1913), and guanine (Lathrop 1912, Schreiner and 
Lathrop 1912). 

3. Lecithin decomposition. A similar instance of a single 
substance decomposing into several substances is that of the 
lecithins. They are closely related to the fats in constitution and 
are possible primary constituents of all plant and animal cells. 
Lecithins are esters of glycerol with two molecules of higher fatty 
acids (palmitic, stearic, oleic acids or other unidentified saturated 
or unsaturated acids) with a molecule of phosphoric acid, which 
is at the same time combined with the base choline. Mathews 
(1915) states that in some cases choline can be replaced by neurine. 
There are a number of different lecithins Avhich are characterized 
by the nature of the organic acid radicals present. The hydrolysis 
may be indicated as follows : 

Ijecithin 



Acids Bases Glyceryl-phosplioric acid 

I I ' I ' 

Palmitic Choline Glycerol 

Stearic or Phosphoric acid 

Oleic Neurine 

or 
Other higher 
fatty acids. 



19 

The base choline is also widely distributed in both plant and 
animal tissues as well as being a decomposition product of lecithins. 
It has been shown to exist in the soil. Choline yields neurine by bac- 
terial decomposition, and both of these compounds break-up into 
trimethylamine. This substance may also be added to the soil from 
other sources, both animal and vegetable. As noted above Stoklasa 
(1911) obtained evidences of lecithin in the soil and Aso (1904) re- 
ports small quantities of lecithin present in soils rich in organic mat- 
ter. Choline has been isolated from soil (Shorey 1913). The tri- 
methylamine reported by Shorey (1913) possibly had its origin 
in the lecithin molecule and it may be that the dihydroxy-stearic 
acid of Schreiner and Shorey (1908 a, 1909 a) and the mono- 
hydroxy stearic acid (Schreiner and Shorey 1908 a, 1909 a) had 
the same origin. (For a discussion of the organic phosphorus of 
the soil see Gortner and Shaw 1917). 

F. Bacterial Processes Which Influence the Form of Soil Nitrogen. 

We know that the decomposition of protein substances can be 
brought about through bacterial activity or by the agency of en- 
zymes widely distributed in the vegetable kingdom. We should 
expect any protein materials present in the soil to be subject to 
the action of the above agencies. Viewed in the light of the 
researches of Emil Fischer (1899-06) protein hydrolysis leads to 
disruption of the complex molecule and the formation of simple 
molecules, as represented in the following- scheme — 

I Di-amino acids 

Proteins Proteoses Peptones Peptides ] Mon-amino acids 

( Acid amides 

Fischer has shown that the amino acid combination in the pro- 
tein molecule may be represented as follows: 

•HO H H O 

H.N-C-C N-C-C-O H 

I I 

R R 

OH 

The gr(jup II I being known as the "peptid group." The 

— C-N— 

nitrogen in this group is in the form of the imino (-NH) radical. 
Upon hydrolysis each "peptid group" takes up a molecule of water 
forming a free carboxyl (-COOH) group changing the imino group 
into an amino (-NH._>| group. 

As the protein hydrolysis continues, the proportion of nitrogen 
in the amino form increases until it reaches a maximum at com- 
plete hydrolysis. It has been shown by Van Slyke (1910, 1911) 
that the amount of amino nitrogen formed is a measure of the 
hydrolysis of the protein substance. The amino acids derived from 
protein degradation may be acted upon by the bacteria in the soil 
and bring about chemical changes which depend largely on the 
character of the organisms present. 



.20 

1. Deamination or reduction plays an important part in the 
formation of ammonium salts in the soil. The amino group is 
split off as ammonia and non-nitrogenous organic acids remain. 
It is not certain whether this process involves oxidation of the 
amino acid to the ketonic acid first, or whether the deamination is 
brought about by hydrolysis. If the hydroxy acids are first formed 
they are subsequently reduced so that the fatty acids are formed 
from the amino acids. This can be illustrated by the following 
examples : 

Aspartic acid will give succinic acid, and this by loss of carbon 
dioxide gives propionic acid. 

Tyrosine — >p-Hydroxy-phenyl-propionic acid — >p-Hydroxy-phenyl-acetic 
acid — >p-Cresol — > Phenol 

This deamination or reduction is in all probability what is 
termed in soil chemistry ammonification. 

2. Decarljoxylation or amine formation involves the splitting 
oft' of carbon dioxide by the action of so-called carboxylase bac- 
teria. This may happen either before or after deamination. Their 
formation is illustrated in the following reactions: 

CH3-CH(NH.)-COOH : :CH:;-CH.-NH., + C02 

Alanine Ethyl amine 

CJT4(OH)-CH.-CH(NH.)-COOH >Cr,TT,(OH)-eH2-GH2-NH2 +CO2 

Tyrosine p-Hydroxy-phenyl-ethyl amine 

NH,-(CH.),-CH ( NH.)-COOH >NIT-CH.-CH.-CH.-CH.-NH.+ C02 

Lysine Cadaverine 

NH^C(NH)-NHCH.-(CH2)^CH(NH2)-COOH > 

Arginine NH.-C(NH)-NH-CH=-(CH2)2-CH2-NH2 + C02 

Agmatine 

Tryptophane gives rise to indole eth^d amine. 

Mathews (1915) states— 

If the splittinar off of carbon clioxide occurs after the deamidization an amine 
cannot, of ccnnse, be formed, but the next lower carboxylic acid is produced 
by way of the aldehyde. 

Thus from tyrosine there may first be formed p-hydroxy- 
piienyl-pyruvic acid, which may be reduced to p-hydroxy-phenyl- 
lactic acid, reabsorbed and recxcreted in the urine ; or the p-hy- 
droxy-phenyl-pyruvic acid may be split into p-hydroxy-phenyl 
acetaldehyde and carbon dioxide, and the former be oxidized into 
p-hydroxy-phenyl-acetic acid, which is excreted in the urine. 

It is well to remember that any bacteria of the coli group will 
split oft' carbon dioxide from an amino acid. It is evident that 
mixed cultures of bacteria may be present in the soil and thus cause 
more than one type of splitting to take place. 

3. Hydrolysis takes place with liberation of carbon dioxide. 
Ehrlich (1911) has shown that yeasts can convert amino acids into 
alcohols, liberating carbon dioxide and ammonia. 

aH4(OH)-CH..-CH(NH2)-COOH + H20 > 

Tyrosine C6H,(OH)-CH2-CH.OH + CO.+ NH= 

Tyrosol 



21 

4. Oxidation results with liberation of carbon dioxide and 
ammonia and the formation of a fatty acid containing- one less car- 
bon atom. A type reaction may be represented as follows: 
R-CH2-CH(NH.)-C00H-+0. >R-CH2-COOH + C02 + NH3 

That oxidation is a factor in the organic matter of the soil 
is self-evident from the fact that carbon dioxide is constantly pres- 
ent in the soil atmosphere in excess of the amount present in the 
air, thus representing degradation of the organic matter to carbon 
dioxide and water, and also from the fact that ammonia is trans- 
formed into nitrates, a process known in soil chemistry as nitrifica- 
tion, a reaction which is carried out in the laboratory by the most 
violent chemical oxidation, e. g., chromic acid. A further step in 
this oxidation carries the nitrates through denitrification which re- 
sults in the liberation of free nitrogen. 

In carrying to completion these processes on protein material 
one can easily postulate an almost unlimited number of organic 
compounds, which are theoretically (and in all probability) pos- 
sible. Very recently Robbins (1916) has produced some evidence 
that the existence of certain of the organic compounds in the soil is 
limited somewhat narrowly by specific bacteria, which either utilize 
the nitrogen or the carbon of the compound as a source of energy. 
Thus pyridine is destroyed by a specific bacterium which is able to 
utilize the nitrogen, and the carbon of cumarin and vanillin is like- 
wise a source of carbon for other specific bacteria. 

G. Nitrogen Distribution in the Soil 

The chemistry of soil nitrogen may to a large extent be con- 
sidered as being the chemistry of protein undergoing- hydrolysis. 
The isolation of a number of amino acids indicates that proteins are 
decomposed in the soil in much the same way as in acicl hydrolysis 
or animal digestion. Just how far the cleavages have already gone 
in the soil previous to acid hydrolysis remains a matter of much 
work before definite conclusions can be drawn. 

Walters (1915) has reported the presence of certain decompo- 
sition products in the soil, presumably proteoses and peptones, 
resulting from either a partial hydrolysis of proteins or by the syn- 
thetic action of microorganisms. It has been recorded by Hoppe- 
Seyler (1909, p. 413) that intermediate protein decomposition prod- 
ucts may result from the action of water at high temperature, by 
mineral acids, alkalies, oxidizing agents, enzymes and microorgan- 
isms. There is little reason to suppose that the action of micro- 
organisms is other than that of the enz^mies which they produce. 
Effront (1914) states that under the influence of the various tryp- 
sins secreted by putrefactive bacteria, the protein molecule is split 
into proteoses, peptones and amino acids. The proteoses and pep- 
tones represent stages of decomposition between that of true pro- 
teins and amino acids. Walters concludes — • 

that proteins undergo hydrolytic decomposition in the soil in much the same 
way as In digestion by enzymes, acids, or alkalies, in the laboratory. 



22 

In an extensive examination of the nitrogen compounds of 
processed fertilizers, Lathrop (1914) has reported the presence of 
certain protein-like substances similar to those described above. 

In his studies on the chemical nature of the organic nitrogen 
in the soil, Jodidi (1911) thoug'ht water ^would be preferable to 
cither acids or alkalies for the purpose of extraction, since it would 
ntit be so liable to alter the organic nitrogenous materials. He 
found that the direct extraction of a soil by boiling with water for 
ten hours removed only 2.92 per cent, and for twenty-four hours 
the highest amount removed from any soil was 9.96 per cent of the 
total soil nitrogen. Shmook (1914), however, reports 19.10 per 
cent of the total nitrogen of a Laterite soil of Russia to be water 
soluble. 

The literature has been very thoroughly summarized by Potter 
and Snyder (1914) in regard to the determination of ammonia in 
soils. Both their work and that of Jodidi (1909) indicates that the 
amount of ammonia is small. Kelley and Thompson (1914) in a 
study of some Hawaiian soils reached the conclusion that ammonia 
and nitrate nitrogen constitute but a small percentage of the total 
nitrogen, and that the nitrogen is very largely in organic combina- 
tion. 

It is known that only a small part of the soil nitrogen is dis- 
solved by dilute acids, yet it has been shown by' Kelley and Thomp- 
son (1914) that 1 per cent hydrochloric acid dissolves some organ- 
ic nitrogen, for in every instance the soils contained only about 
half as much ammonia nitrogen as was extracted by the acid. 

In the soil studies of Potter and Snyder (1915 a) they find that 
the nitrogen extracted by 1 per cent hydrochloric acid varied from 
about 1.2 to 2.3 per cent oi the total nitrogen, except in the case of 
the peat it was only 0.67 per cent. This is contrary to the findings 
of (iortner (1916a). AVorking with eight mineral soils he finds a 
maximum of 4.18 per cent of the total nitrogen soluble in 1 per 
cent hydrochloric acid with an average of 3.17 per cent. In three 
peats he finds a maximum of 7.50 per cent with an average of 3.78 
per cent, and in five samples of unchanged vegetable materials (oat 
straw, alfalfa hay, oak leayes. sweet fern leaves, and grass from a 
peat bog) he finds a maxirnum of 34.58 per cent with an average of 
20.10 per cent. These findings would seem to indicate that in the 
transformation of vegetable materials into the true organic mat- 
ter of the soil there is a fall in the proportion of the total nitrogen 
soluble in very dilute acids. 

Shorey (1905) published resvilts of his investigations which 
gave the first definite knowledge of the individual amino acids 
formed in the decomposition of soil organic matter. He worked on 
a Hawaiian soil with a view to classifying the decomposition prod- 
ucts of the nitrogenous substances in the soil. The method applied 
was that proposed by Osborne and Harris (1903) for classifying the 
decomposition products of proteins resulting- from acid hydrolysis. 
The method is a modification of that proposed by Hausmann (1899) 
and is in short as follows : After hydrolysis the excess of the 



23 

mineral acid is removed by evaporation, and the nitrogen present 
as ammonia determined by distilling with an excess of magnesium 
oxide; after separating the magnesia precipitate from the remain- 
ing solution by filtration, the nitrogen was determined in the pre- 
cipitate by the Kjeldahl method, the di-amino nitrogen in the fil- 
trate was precipitated by phosphotungstic acid and determined by 
the method of Kjeldahl and the mon-amino nitrogen determined 
by difference. 

He obtained in the acid solution 84.5 per cent of the total nitro- 
gen in the soil, 52.3 per cent of which was found in the magnesia 
precipitate. This result is in striking contrast to those obtained by 
Osborne and Harris (1903) working on pure proteins, where they 
found that the nrtrogen contained in the magnesia precipitate does 
not usually exceed 4 per cent of the total nitrogen and in most 
cases is very much less. The amount of nitrogen insoluble in the 
12 per cent acids used in the digestion may be designated as ''hu- 
min." The nitrogen in the magnesia precipitate has been desig- 
nated by most investigators "humin" nitrogen. The total "humin" 
nitrogen in the soil is then represented by the nitrogen in the 
magnesia precipitate plus that retained by the soil. On recalcula- 
tion of his data it was found that the insoluble humin in the soil 
after hydrolysis amounted to 15.3 per cent of the total nitrogen, 
making a total humin nitrogen content of 59.1 per cent. This very 
high result of total humin nitrogen was undoubtedly due to the soil 
being hydrolyzed only seven hours with a relatively low concen- 
tration of hydrochloric acid and the insoluble residue boiled the 
same length of time with sulfuric acid. Complete decomposi- 
tion of the proteins probably did rrot take place in the dilute acids 
used in the short time that they were heated. As a result partially 
hydrolyzed residues may have been precipitated by the magnesium 
oxide, which would account for the high results. 

Shorey (1906) concluded that even though we might know 
much concerning the constitution of the compounds comprising 
the various groups isolated from protein by this method of analysis, 
we know nothing concerning their composition when isolated from 
soil, inasmuch as we are not dealing with a pure protein (cf. also 
Gortner 1913, 1914, 1916 c). 

The work of Suzuki (1906-08 c) gives us further knowledge of 
the individual amino compounds formed in the decomposition of soil 
organic matter. He worked with three samples of humic acid, one 
obtained from Merck (origin unknown to Suzuki), one prepared 
from an unmanured soil, and one from a compost heap. After 
boiling each preparation for ten hours with strong hydrochloric 
acid, the undecomposed residue was filtered off, washed, and the 
residue extracted twice in this manner with strong h3^drochloric 
acid. He determined the amounts dissolved as amide, di-amino, and 
mon-amino acid nitrogen. From 65 to 75 per cent of the total 
nitrogen Avas dissolved by the hydrochloric acid and in the extract 
41 to 62 per cent of the nitrogen was not precipitated by phospho- 
tungstic acid. A sample of humic acid was twice extracted, with 



24 



concentrated acid and the residue analyzed. His results calculated 
on the ash free basis showed the residue to contain 64.11 per cent 
carbon, 3.35 per cent hydrogen, and 0.80 per cent nitrogen. The 
residue becomes lower in nitrogen*, hydrogen, and ash but richer 
in carbon as the hydrolysis is continued. 

Detmer (1871) pointed out that similar results were true in 
peat beds where the deposits remained undisturbed for years. He 
found that there is an increasing carbon and nitrogen content of 
the humus for varying depths. This is shown by the followins; 
table: 



Carbon 



Brown peat, near the surface.. 

Dark peat, 7 feet 

Black peat, 14 feet 



57.75 
62.02 
64.07 



Hydrogen] Oxygen 



5.43 
5.21 
5.01 



36.02 
30.67 
26.87 



Nitrogen 



0.80 
2.10 
4.05 



Likewise Gortner (1917) observed — 

that there is a much greater wastage of carbon than nitrogen. Hilgard 
(1906) calls attention to the increased nitrogen content of the humus over 
that of the original vegetable materials. If we take the average carbon con- 
tent of proteins as 51.15 per cent (average of .^0 analyses given by Mathews 
1915) a C:N ratio of 3.06 found in soil A-1916 would give a nitrogen content 
of 16.71 per cent, which approaches very nearly to the average nitrogen content 
of these 30 proteins, i. e., 17.66 per cent. It is evident that the materials re- 
maining in the soils are rapidly increasing in nitrogen content. 

Suzuki (1906-08 c) made further studies on a 500 gram sample 
of the humic acid obtained from Merck. It was hydrolyzed with 
concentrated acid and the solution obtained subjected to esterifica- 
tion and fractional distillation according to the method of Fischer 
(1901). He obtained: 

Alanine 2.39 gm. 

Leucine 2.16 gm. 

Alanine + aminovalcrianic acid • • ■-..., 0.11 gm. 

Aminovalerianic acid 0.57 gm. 

Proline ( copper salt of active proline) . 0.67 gm. 

(copper salt of inactive proline) (?) 0.50_ gm. 

Aspartic acid 0.06 gm. 

Impure aspartic acid (?) . 3.16 gm. 

Glutaminic acid present 

Tyrosine -. .trace 

Histidine trace 

Ammonia 1 .90 gm. 

Copper salts of unknown acids 30.30 gm. 

As these compounds are typical protein decomposition prod- 
ucts, his work proves that the humic acid examined by him was 
either of a protein nature, a mixture of protein decomposition prod- 
ucts, or probably both together with some as yet unknown com- 
pounds. Unfortunately, the origin of the acid was unknown to 
Suzuki, but he states that it was probably prepared from peat. 

From a study on Michigan peat soils Jodidi (1909) has con- 
cluded that the bulk of the organic nitrogen is made up of acid 
amides, di-amino acids, and mon-amino acids. He used slightly 
modified methods. The ammonia was determined as above by 
distillation with magnesium oxide. The residue from distillation 
with magnesia was dissolved in dilute sulfuric acid and the 

*He stated that although the nitrogen content decreases, it is very difRcult 
to entirely remove all of the nitrogen. 



25 

di-amino acids precipitated by phosphotungstic acid. The nitrogen 
in the precipitate of di-amino acids was determined by the method 
of Kjeldahl. The filtrate from the di-amino acids containing the 
mon-amino acids was oxidized by the Kjeldahl method and the 
nitrogen determined. In some cases he secured the mon-amino 
nitrogen by difiference, stating that it was difficult to get a direct 
deterpiination of the mon-amino nitrogen by the Kjeldahl method. 
He states that — 

this percentage was usually higher than the one directly found by kjeldahl- 
i'/Ang the filtrate from the phosphotungistic acid precipitate. 

In one experiment the percentage of mon-aminOi nitrogen by 
direct determination was 62.83 per cent, while by difference the 
result was 67.22, and in another case the results were 64.25 and 
65.06 respectively. 

It will be noted that this is a departure from the method used 
by Shorey (1905) in that here the nitrogen is separated into three 
fractions instead of the usual four. The nitrogen in the magnesia 
precipitate was distributed with the di-amino and mon-amino acid 
nitrogen. This method of nitrogen distribution will be classed 
as "Jodidi numbers" (in contrast to the Hausmann numbers) in 
the subsequent portion of the paper. 

Van Slyke's (1910) nitrous acid method was first applied by 
Robinson (1911) to a study of peat soil, in order to determine the 
amount of amino nitrogen present. The ammonia nitrogen was re- 
moved by previous distillation with magnesium oxide. The only 
value of Robinson's work seems to be in the fact that his figures 
for total and amino nitrogen increase to a maximum with in- 
creasing time of hydrolysis, in much the same manner that pro- 
teins react ; thus indicating that the amino groups were not ex- 
isting free in the peat but in some form of combination which 
did not react with nitrous acid. For example, after one hour's 
hydrolysis the total nitrogen of the soil in solution amounted to 
29.86 per cent, while the amino nitrogen was 4.62 per cent or a 
ratio ex'ceeding 6:1. After forty-two hours' hydrolysis the nitro- 
gen of the soil in solution was 51.54 per cent of the total nitrogen 
and the amino nitrogen was 25.07 or a ratio only slightly exceed- 
ing 2:1. This ratio increases again with further hydrolysis so 
that at the end of 138 hours the ratio is almost 3:1. However, the 
amount of nitrog"en in solution was so> small that the experimental 
error of measuring total and amino nitrogen must have been 
quite large. 

A-Iore recently Jodidi (1911) made a study of some Iowa soils 
using a modification of the Osborne and Harris (1903) method. 
The nitrogen removed from the solution by the magnesium oxide 
was ignored by the author.* " This contained a part of the so-called 
"humin" nitrogen. Subtracting the sum of the ammonia** and 
di-amino nitrogen from 100 he found the per cent of the nitrogen in 

*Experimental data presented later in this paper will show that this 
fraction may exceed 9 per cent of the total nitrogen. 

**He distinguishes the ammonia nitrogen originally present in the soil 
as such, from that produced by acid hydrolysis. 



26 

solution as mon-amino nitrogen. It will be readily seen this con- 
clnsion is incorrect. The mon-amino acid nitrogen as deter- 
mined represents the sum of the humin and mon-amino nitrogen. 
It is very unfortunate that this mistake should have been made 
since this gives us only the actual ammonia and di-amino acid 
nitrogen for use in comparison with other investigations of the 
organic soil nitrogen as distributed by acid hydrolysis. Kelley 
(1914) following details as outlined by Jodidi (1911) has made 
the same error and the criticisms above apply with equal force to 
his data. 

It is also extremel}^ ttnfortunate that investigators should in 
any case rely upon figures obtained "by difiference" for any one of 
their fractions. It is sometimes permissible to use figures obtained 
in this manner, for example, in case a determination has been lost 
and lack of time or other consideration prevents a repetition; 
but to constantly use figures obtained in this manner is unscientific, 
especially, since by this method we have no means of determining 
how great the experimental error of the method may have been. 

Jodidi (1911) called attention to the fact that in the case of 
protein substances the distillation of the hydrolyzed protein with 
magnesium oxide gives pure ammonia. This, however, may not 
hold true for the hydrolyzed portion of soils, since some protein 
substances through decay yield organic bases. It has been shown 
by Bocklisch ( 1885) that dimethyl amine is formed through putre- 
faction of fish, and trimethylamine has been produced by the 
])Utrefaction of wheat flour and fish. The bases putrescine and 
cadaverinc result from the decay of organic substances under cer- 
tain conditions. It is possible for certain di-amino acids to be- 
come transformed into di-amines, as for example, arginine can be 
decomposed into urea and ornithine through bacterial activity. 
These processes can be expressed by the following equations : 

NH,-C(NH)-NH-CH.--(CH=)2-CH(NH.)-COOH + H.O^^> 

Arginine 

NH. 

NH2-CH2-CH2-CH.-CH (NH.)-COOHH-C=0 

I 
Ornithine NH; 

Urea 
NH.-CH.-CH.-CH.-CH(NH2)-COOH > 

C)rnithine -, 

CO.+ NH..-CH.-CH2-CH.-CH.-NH2 
Putrescine 

Jodidi found that the ammonia ol:)taincd by distilling the 
evaporated extract of the soil with magnesium oxide was actually 
pure ammonia, thereby establishing the absence of any volatile 
organic bases ; but that the phosphotungstic acid precipitate and the 
filtrate from that precipitate did not represent di-amino and mon- 
amino acids only. 

In order to find out how much of the di-amino and mon-amino 
nitrogen actually belonged to di-amino and mon-amino acids, the 
solutions were subjected to analysis by the formaldehyde-titration 



27 

method of Schiff (1900. 1901, 1902) as modified by S5rensen (1908), 
Henriques (1909), and Henriques and Sorensen (1910). 

In a comparison of the amount of di-amino* acid nitrogen, 
calculated as if histidine. arginine, and lysine were present in about 
equal amounts, he finds that in plot E, 101.8 per cent; plot O, 84.8 
per cent; and plot U, 93.9 per cent of the nitrogen in the phos- 
photungstate fraction was actually present as di-amino acids. 

However, he obtains widely divergent results for mon-amino 
acid nitrogen; in plot E, 91.64 per cent; plot Q, 52.63 per cent; 
plot U, 40.12 per cent; plot H, 88.31 per cent; and plot J, 92.11 
per cent of the total nitrogen in the "filtrate from the bases" was 
actually present as mon-amino acid nitrogen whereas all should 
have been present in this form if dealing with pure proteins only. 
He, therefore, concludes that the di-amino and mon-amino acids, 
or in other words, the bases and filtrate from the bases by hydro- 
lyzing soils, contain other products than are formed by hydrolysis 
of pure proteins. 

' In a series of fertilized soils studied by Lathrop and Brown 
(1911) they find that almost 98 per cent of the nitrogen in the soil 
is of organic nature. The ammonia and nitrate nitrogen constitute 
the remainder. Employing the same method to the distribution of 
the soil organic nitrogen as Shorey (1905), they boiled 100 grams of 
soil with 500 cc. of hydrochloric acid (sp. gr. 1.115) for three hours, 
and used the filtrate after making to definite volume for the 
analyses. The figures given for ammonia nitrogen represent the 
actual amount of nitrogen as ammonia obtained by hydrolysis and 
does not include the ammonia nitrogen already present in the 
soil. They find that the plots which have received organic fer- 
tilizers give the largest amount of ammonia on hydrolysis, the 
amount being highest in the plot which has received manure alone 
and lowest in the check plot. 

Of the five soils studied, four contained, over 25 per cent of 
the "humin" nitrogen soluble in acid, while the other only showed 
about half as much. Since the nitrogen of the soil not soluble in 
acid may be considered "humin" nitrogen, the total amount in this 
form in the above four soils was over 53 per cent, while in the 
other soil, which received dried blood, it aniounted to only 43 per 
cent. 

However, the fractions which they determined have actually 
very little significance in a discussion of protein hydrolysis products, 
inasmuch as a three-hour hydrolysis is far too short a time to com- 
pletely decompose the protein molecule. This explains their high 
figures for humin nitrogen and low ones for mon-amino acid 
nitrogen. The di-amino and mon-amino^ acid nitrogen dififer rather 
widely but there seems to be no agreement between the form of 
nitrogen and the plot treatment. 



*The exact interpretation of his data is difficult to understand. 



28 



In conclusion they say- 



these five samples of soil are really the same soil under long- continued 
treatment of different kinds. It is not improbable that work on widely differ- 
ent soils will show even much greater variations than those here noted. 
The work shows, however, that even in such cases there is a difference in 
the nitrogenous compounds in the soil, and that different decompositions of 
the nitrogenous matter has taken place and probably will continue to take 
place, under the different conditions imposed upon the soils in the field. 

A very interesting- study has been made by Shmook (1914) 
of the nitrogen distribution in four Russian soils, one of the Podzol 
type, two of the Chernozem type, and one of the Laterite type by 
applying the method of Hausmann (1899). The water extract 
from 100 grams of the Podzol soil showed a very high content of 
soluble nitrogenous compounds. This amounted to 0.0452 gram 
of nitrogen which constituted 19.10 per cent of the total soil. nitro- 
gen. This was distributed as follows: amide nitrogen 0.0034 
gram, di-amino nitrogen traces, and mon-amino nitrogem 0.0408 
gram. These results were deducted from the analyses of the 
hydrolyzed soil. Thirty gram samples of soil were hydrolyzed 
with 120 cc. of h3''drochloric acid (sp. gr. 1.12) for 8 hours and 
the analysis carried out as directed. 

He finds, that the Chernozem and Podzol soils show a simi- 
larity in the distribution of amide nitrogen, and that of the amino 
acid nitrogen, but that the nitrogen distribution in the Laterite 
soil is entirely different from the other types, tie concludes that 
the amount of protein in the soil is not in direct relation to the 
amount of organic matter, and that the nitrogen insoluble in hydro- 
chloric acid occurs in unknoAvn form and composes only 1.50 to 
1.90 per cent of the organic matter of the Chernozem and Podzol 
soils, but 13.70 per cent of the total organic matter of the Laterite 
soil, after subtraction of the protein nitrogen belonging' to this 
insoluble portion. He suggests that these results would indicate 
that the organic nitrogen existed in the soil in large part as protein 
material in the Chernozem and Podzol soils, but that a considerable 
portion was of a non-protein origin in the Laterite soil, since 
the amount of this insoluble "melanin" in pure proteins amounts 
to from 0.60 to 1.80 per cent of the total protein nitrogen.* 

Potter and Snyder (1915 a) made a study of some Iowa soils 
using Van Slyke's (1911) method of protein analysis. Their soils 
were the same type but had received different fertilizer treatment. 
At the same time they also made a study of peat soil. The soils 
were in all cases first extracted with 1 per cent hydrochloric acid 
"in order to render the humus more soluble." They were then 
hydrolyzed by boiling one part of the soil with two parts of 22 
per cent hydrochloric acid for forty-eight hours. They also pre- 
pared a 1 per cent sodium hydroxide extract of the acid leached 
soils, and after precipitation with sulfuric and acetic acids the 
resulting humic acid precipitate was subjected to the above method 
of analysis. 

The authors conclude: (1) that the humiii nitrog'en as deter-. 
mined by the Van Slyke method on soils extracted by dilute alkali 

*Actually in some cases these results are much lower, and in others de- 
cidedly Higher, e. e., Van Slyke (1911) finds gelatin contains 0.07 per cent and 
fibrin 3.17 per cent. 



29 

IS very. high when compared to the amounts in proteins; (2) that 
no typical class of organic compounds is extracted from the soil 
by dilute alkali ; (3) that the amounts of amino acid and peptid 
nitrogen in the soil are found to be very small compared to the 
amounts of amino acids formed by acid hydrolysis ; (4) (and this 
is the most important for our purpose) that 

nothing very significant can be deduced from the variations in the different 
soils, 

or in other words, the organic matter in the same soil type under 
different fertilizer treatment; is essentially the same, and as I shall 
show later in the experimental part of this paper, the organic mat- 
ter (as distributed by Van Slyke's method) in different soil types is 
essentially the same. 

Lathrop (1916) recently made a study of protein decomposi- 
tion in the soil. He added a high grade nitrogenous fertilizer to 
the soil and allowed decomposition to proceed at laboratory tem- 
perature, and at different periods took samples and subjected them 
to Van Slyke's method of protein analysis in order to determine 
how the different fractions were aff"ected by bacteria and other 
agencies present in the soil. 

From h[s work he concludes that the analysis obtained by the 
Van Slyke method indicates that there is a formation of protein 
taking place in the soil in the course of the decomposition of the 
protein materials, and that apparently the new protein is somewhat 
resistant to decomposition. He states, that — 

this is indicated in (1) the unequal loss of mon-amino acids and hydrolyzable 
nitrogen from the soil during the early stages, (2) by an increase in amide 
nitrogen during the early stages, (3) by an increase in histidine nitrogen dur- 
ing the early stages, (4) by an increase in the arginine nitrogen during the 
later stages, and (5) by an increase in lysine nitrog-en during the later stages. 

This view that the protein nitrogen in the soil was largely con- 
tained in the bodies of bacteria and protozoa had been previously 
advanced by. Shmook (1914). 

It was stated by Loew and Aso (1906-08) that under favor- 
able condition of growth protein material is excreted by yeast and 
bacteria, and that soluble materials can pass through the cytoplasm 
to the outside on death of the cell. They also state that the amount 
of nitrogenous substances partly consisting of peptones excreted 
by 'dead cells, is by no means inconsiderable. 

H. A Summary of the Nature of the Organic Matter of the Soil in 
the Light of Our Present Knowledge. 

It has been pointed out, that the organic matter of the soil 
was at first considered to be a very simple thing, that the alkali 
extract contained the essential plant nutrients, and that the process 
of "humification" was the necessary step through which the or- 
ganic matter of the oil must pass in order to be converted into fo.od 
materials for plant life ; but as the knowledge of chemistry de- 
veloped it became evident that the problem was more complex. 
A definite knowledge of the forms of organic matter in the soil 
can only be secured when we have a thorough understanding of 



30 

the chemical products formed by the action of the bacteria, pro- 
tozoa, and fungi on each other, and on the organic matter, both 
animal and plant, that finds its way to the soil. The present method 
is to study the soil with its complex mixture of organic and in- 
organic compounds, and by the application of recognized methods 
of chemical investigation unravel the mysteries tied up in it; 
but unless we have the climatic and cultural conditions uniform, 
we cannot hope to secure results that will be of general application. 

There have been two methods of attacking the problem : first,, 
the isolation and study of the individual organic compounds ex- 
isting in the soil, and, second, a study of the hjiirolysis products. 
It is evident that these methods are slow and tedious and unsatis- 
factory, but on the other hand a study of all the possible combina- 
tions of the organic substances existing in the soil appears to be 
an endless task in the light of our present knowledge. In brief, 
a complete and thorough knowledge of the organic matter -of the 
soil appears possible only after we have mastered all of the bio- 
chemical processes which are characteristic of the fungi, protozoa, 
and bacteria of the soil, and have a much deeper insight into 
the chemical constitution of vegetable cells than we have at the 
present time. (3ur present knowledge leads us to believe that 
it is possible to isolate an almost infinite variety of chemical 
compounds from a soil, the number and variety reaching a limit 
only when we have isolated all of the compounds which are pres- 
ent in the plants which grew upon the soil, plus those compounds 
contained in the bodies of bacteria, protozoa, and fungi, plus all of 
the compounds which may be derived from these compounds 
under the peculiar soil conditions of decay, oxidation, bacterial ac- 
tion, and the secretions of fungi and living plant roots. 



31 



II. EXPERIMENTAL: A STUDY OF THE NITROGEN 
DISTRIBUTION IN DIFFERENT SOIL TYPES. 

A. The Problem. 

It has been shown in the historical study above that a number 
of investigators have studied the organic nitrogen distribution in 
the soil by applying either Hausmann's(1899) or Van Slyke's (1910, 
1911) method of protein analysis. It has been demonstrated by 
Potter and Snyder that various plots on a single soil type under 
different fertilizer treatments gave, with Van Slyke's method, es- 
sentially the same results. 

I have taken up the problem at this point and made a similar 
study of the organic nitrogen distribution in different soil types in 
an attempt to see whether the forms in which nitrogen occur 
differ from locality to locality and from soil type to soil type. 1 
am concerned with the problem of distribution of the organic nitro- 
gen in the soils and soil extracts studied. 

B. The Material. 

The study was made using two peats, one muck, seven mineral 
surface soils, and one subsoil. All were from the State of Min- 
nesota. The origin and type names are in accordance with the 
surveys of the Bureau of Soils of the U. S. Department of Agri- 
culture when such surveys were available. Almost all of the soils 
used are portions of the identical samples employed by Gortner 
(1916 a, and 1917) in his soil studies. The samples used in this 
study were all air dry soils. The moisture was determined by 
heating the soils to a temperature of 100° C. for 12 hours and then 
weighing-. 

The descriptions of the soils follow : 

L Calcareous black grass-peat. This sample was selected 
from a large bulk sample taken to a depth of 8 inches from a 
grass bog near the Agricultural Experiment Station Farm, St. Paul. 
The peat was black and well decomposed. The peat was g-round 
to a powder in a ball mill before using. The air dry material con- 
tained 6.40 per cent moisture. 

2. Sphagnum-covered peat. This sample of very strongly acid 
peat was prepared from a large bulk sample taken from a bog on 
the Experimental Farm near Grand Rapids. The superficial layer 
of sphagnum and shrubs was first removed and a sample of the un- 
derlying peat taken to a depth of 8 inches. The peat was poorly 
decomposed. The sample was prepared by grinding to^ a powder in 
a ball mill. The air dry soil contained 5.90 per cent moisture. 

3. Acid "muck" soil. A sample of this strongly acid soil was 
obtained from a bosf about two miles from the farm of the Agri- 



32 

cultural Experiment Station, St. Paul. The sample is a composite 
of 10 samples taken to a depth of 8 inches lengthwise of the bog. 
The vegetation of the bog was largely cattails (Typha spp.) and 
rushes (Scirpiis spp.) The sample contained 5.60 per cent moisture 
in the air dry condition. ^ 

4. Fargo clay loam. The sample analyzed consisted of a 
composite made from 144 borings taken to a depth of 8 inches from 
a twenty-acre field on the Northwest Sub-station Farm, Crookston. 
The sample was highly calcareous. This soil type has been de- 
scribed by Mangum and Schroeder (1906). The moisture content 
of the air dry soil was 3.92 per cent. 

5. Fargo silt loam. This sample was a composite made from 
100 borings to a depth of 6 inches, twenty borings being taken from 
each of five virgin fields near Nerstrand, Rice County. The sam- 
]:»le had a neutral reaction. The soil type has been described by 
Burke and Kolbe (1909). The air dry soil contained 14.89 per cent 
moisture. 

6. Carrington silt loam. This soil is represented by two sam- 
ples. Each composite consisted of 100 borings to a depth of 6 
inches, twenty borings being taken from each of five virgin fields 
near Nerstrand, Rice County, for Sample number I, and twenty 
borings being taken from each of five virgin fields near Morristown, 
Rice County, for Sample number IL Sample I was strongly acid 
while Sample II was but slightly acid. The soil type is described 
by Burke and Kolbe (1909). The moisture content of sample I was 
6.22 per cent. 

7. Hempstead silt loam. A composite sample from 36 borings 
to a depth of 6 inches was taken from 12 plots on the Agricultural 
Experiment vStation Farm, St. Paul. No commercial fertilizer had 
been applied, but the land had long been under cultivation. The 
soil type has been described by Smith and Kirk (1914). The, sam- 
ple was strongly acid. The air dry soil contained 3.07 per cent 
moisture. 

8. Prairie-covered loess. The sample consisted of 50 borings 
taken to a depth of one foot, ten borings being taken from each of 
five virgin fields near Luverne, Rock County. The sample was 
somewhat calcareous. The area from which the sample was ob- 
tained has not been subjected to a detailed soil survey. The air 
dry soil contained 7.89 per cent -moisture. 

9. Forest-covered loess. This sample was taken from five 
virgin fields near Caledonia, Houston County, ten borings to a 
depth of one foot being taken from each field and ec[ual weights 
from each boring being combined in the composite sample. The 
sample was strongly acid. The air dry soil had a moisture content 
of 1.87 per cent. 

10. Hempstead silt loam subsoil. This was a third foot bulk 
sample taken from a gro^'c on the farm of the Agricultural Experi- 
ment Station, St. Paul. The sample was strongly acid. 



33 
C. The Method. 

The method of Van Slyke (1911) has been used throughout 
this investigation because the nitrogen can be separated into a 
larger number of fractions than when the earlier method of Haus- 
mann (1899) is employed. The different fractions, however, are 
not listed in the same manner as in the Van Slyke method, for 
since we are not dealing with pure protein material we cannot 
correctly speak of arginine, histidine, cystine, and lysine nitrogen. 

Van Slyke (1915) has called attention to the fact that his 
method was devised for the analysis of pure protein material and 
not for a heterogeneous mixture of nitrogen compounds. This 
fact was not recognized by certain investigators. Grindley and 
Slater (1915) applied this method to the anal3^sis oi feeding stuffs 
in exactly the same manner as though they were dealing with a 
pure protein. Potter and Snyder (1915 a) analyzed certain soils 
and soil extracts and report their fractions as "arginine," "histi- 
dine," etc. Although they state (p. 2221) : 

It is not thought that nitrogen as found by the Van Slyke method, work- 
ing with such a complex as the soil, is in reality, all lysine, histidine, etc., 
nitrogen. It might be said that each group, as found, represents a class ,of 
compounds having the particular reaction by which the lysine, histidine, etc., 
nitrogen respectively are determined. 

It is obvious that there are other types of organic materials 
which will interfere with the nitrogen distribution. It seems very 
probable that in soils as well as in the material analyzed by Grind- 
ley and Slater (1915) there must be many organic nitrogen com.- 
pounds which have no relation to the protein molecule, such as 
purine bases, pyrimidine bases, nitrogenous lipins, nitrogenous pig- 
ments, as well as other non-protein nitrogenous compounds (cf. 
the list of non-protein nitrogenous compounds actually isolated 
from the soil as given in the preceding part of this paper). Gortner 
(1913) states that much valuable comparative data can be obtained 
by the application of Van Slyke's method to the analysis of heter- 
ogeneous materials ; but it is self evident that no analogy can be 
drawn between the analysis of pure protein and the analysis of a 
protein mixed with an unknown amount of foreign nitrogenous 
compounds. The results of Potter and Snyder (1915 a) are of 
little value in advancing our knowledge of soil proteins or for 
comparison with analyses of pure proteins, but are extremely 
valuable and interesting for comparison between themselves and 
with other analyses of soils carried out under similar conditions. 
It must be remembered that all data on similar material is strictly 
comparable when the same method of analysis is followed. 

It is possible that many of the non-protein nitrogenous com- 
pounds may be split up during the hydrolysis of heterogeneous ma- 
terial. Gortner (1913) has shown that uric acid nitrogen is dis- 
tributed in all four of the major fractions after hydrolysis. The 
ammonia nitrogen amounted to 15.27 per cent, humin nitrogen 
35.98 per cent, basic nitrogen 12.97 per cent, and non-basic nitrogen 
35.78 per cent. "The humin nitrogen contained no trace of black 
color and was probably calcium ureate." Probably all of the purines 
and pyrimidines would behave in a similar manner. 



34 

The general method employed in this investigation will be dis- 
cussed in detail for two soils, a peat and a mineral soil, inasmuch as 
the experimental conditions vary in minor details with the two 
types. 

1, The method in detail for a peat soil. Duplicate samples 
of 15 grams were hydrolyzcd in the presence of hydrochloric acid 
for 48 hours. The content of calcium oxide was taken into account, 
and corrections made so that the 100 cc. of hydrochloric acid used 
was of sufficient concentration to neutralize the lime and at the 
same time furnish a constant boiling acid. The hydrolysis was 
carried out in 200 cc. long neck, round bottom Kjeldahl flasks, fitted 
with modified Hopkins condensers made from a test tube which 
fit rather loosely into the neck of the flask. By means of this device 
any error due to products extracted from cork or rubber stoppers 
was obviated. The flasks were heated to gentle boiling on the 
same sand bath over an Argand burner, so that the rate of hydro- 
lysis would be as near the same as possible. 

After completion of the 48 hour hydrolysis the mixture was 
evaporated in a Claissen distilling flask under diminished pressure 
until all the hydrochloric acid possible was driven oft'. The residue 
after this distillation was dissolved in 100-150 cc. of water, 100 cc. 
of 95 i)er cent alcohol, and an excess of calcium hydroxide sus- 
pended in water was added and the ammonia distilled off into 
standard acid at a temperature of 40-50° C. under a pressure of less 
than 30 mm., distillation being continued for at least a half hour. 
The results are listed under "ammonia nitrogen." 

The alkaline mixture in the distilling flask was filtered and 
the precipitate well washed with hot water until free of chlorides. 
A Kjeldahl determination was made of the filter and its contents, 
and the results listed under "humin nitrogen." 

The filtrate and washings from the humin were acidified with 
hydrochloric acid and concentrated under diminished pressure to 
less than 200 cc. 1Y> this solution Avas added 18 cc. of concen- 
trated hydrochloric acid and the whole heated on the water bath 
until hot. A solution containing 15 grams of phosphotungstic acid 
was then added and the heating on the water bath continued for 
an hour. The flask was then set aside in a cool place for 48 hours. 
The precipitate of the bases was then filtered off and washed as 
directed by Van Slyke (1911). 

The basic phosphotungstates were suspended in 800 cc. of 
water and brought into solution by the cautious addition of a 50 
per cent solution of sodium hydroxide, a few drops of phenolph- 
thalein being added to guard against too great an excess of alkali. 
The phophotungstic acid was precipitated by the addition of a 
slight excess of 20 per cent barium chloride, and the barium phos- 
photungstate was filtered off and washed free of chlorides with 
hot water. 

The filtrate and v/ashings Avere united, acidified with hydro- 
chloric acid, and concentrated under diminished pressure to a ^ery 
small volume. After cooling the residue was filtered, washed and 
made up to 50 cc. volume. 



35 

The washed precipitate of barium phosphotungstate and filter 
were subjected to Kjeldahl determination for any nitrogen that 
might be held by absorption, adsorption, or occlusion, as was also 
the filter and contents remaining after the final filtration of the 
solution containing the basic nitrogen. In all cases some nitrogen 
was found. This nitrogen is probably de-ived from the "unad- 
sorbed humin carried down with the l^asic phosphotungstates" 
mentioned by Van Slyke (1915, p. 284). Inasmuch as my work was 
done prior to this publication, I added this nitrogen t(^ the total 
nitrogen content of the bases instead of to the humin. 

During the distillation after kjeldahling this precipitate the 
cochineal indicator took on a color which made the acid solution 
appear that complete neutralization might have occurred when in 
fact it had not. This color change was noticed in every case with 
the barium phosphotungstate distillate. This made titration difti- 
cult, since a new end point had to be established. Gortner and 
Holm (private communication) have observed a similar color 
change in the case of fibrin hydrolyzed in the presence of a large 
excess of formaldehyde. They explain this finding on the assump- 
tion that pyridine (or some similar base) is formed which is not 
easily broken down in the Kjedahl process (cf. Dakin and 
Dudley. 1914), and which -greatly influences the color changes of 
the indicator when the base is volatalized during the subsequent 
distillation with alkali. Whether or not this is the cause of the 
phenomenon observed in my materials cannot be ascertained with- 
out further investigation. 

In no case did I attempt to separate the basic nitrogen into 
the usual fractions of "arginine." "cystine," "histidine," and "lysine" 
nitrogen, because I am not dealing with pure protein. Instead in 
each case the total nitrogen liberated as ammonia was determined 
on 25 cc. of the solution containing the bases. This was determined 
in exactly the same manner that Van Slyke used for the determina- 
tion of arginine iiitrogcn. The volume of standard acid neutralized 
by the ammonia indicated the amount contained in the 25 cc. of 
solution used. The nitrogen found is listed as "basic nitrogen set 
free as ammonia by 50 per cent potassium hydroxide." The solution 
remaining- from this determination was used in the estimation of 
the total nitrogen of the bases. This Avas performed according to 
Van Slyke's directions. The quantity of acid neutralized in this 
determination, was added to that neutralized in the basic-nitrogen- 
sct-free-as-ammonia-by-50-per-cent-potassium-hydroxide. thus se- 
curing the "total basic nitrogen." 

The "amino nitrogen of the bases" was determined in Van 
Slyke's (1912) apparatus, using 10 cc. portions of the solution. 

The filtrate from the bases was treated with sodium hydroxide 
solution until a slight turbid precipitate of lime Avas formed, and 
then cleared immediately by the addition of acetic acid. This was 
concentrated under diminished pressure and on cooling- was made 
to 200 cc. volume. The solutions were more or less violet in color. 
"Total nitrogen in the filtrate from the bases" was determined on 
duplicate portions of 25 cc. each by the method of Kjeldahl. The 



36 

digestion was continued for three hours after the solutions were 
clear, so that the phosphotungstic acid would not interfere with 
the accuracy of the determination. The "amino nitrogen in the 
filtrate from the bases" was determined on duplicate portions of 
10 cc. each by means of the \'an Slyke (1912) apparatus. 

2. The method in detail for a mineral soil. Duplicate portions 
of 250 grams were hydrolyzcd in 500 cc. round bottom Kjeldahl 
flasks for 48 hours on dififerent sand baths. Allowance was always 
made for the lime content of the soil, and the requisite amount of 
hydrochloric acid added to insure the presence of a constant boiling- 
acid (sp. gr. 1.115), and a volume of approximately 250 cc. The 
solutions boiled smoothly and gave no trouble by bumping. Air 
dry soil was used in all cases, but the moisture was determined on 
a separate portion and all data calculated to the dry basis. 

On completion of the hydrolysis each of the two samples was 
diluted to a 1000 cc. in measuring flasks and allowed to settle for 
at least 24 hours. The clear solution was then isyphoned off and 
an aliquot of 500 cc. analyzed according to the usual method of 
Van Slyke. In nearly all cases this solution was straw color due 
to the presence of ferric salts that had been formed during the 
kydrolysis. No black color, the usual color of a protein hydrolysate, 
Avas observed in any instance. 

Another aliquot of 100 cc. was used for the determination of 
total nitrogen in the solution by making duplicate Kjeldahl de- 
terminations on 50 cc. portions. A second aliquot of 100 cc. was 
used for the determination of "Jodidi numbers" (cf. p. 25) when 
they were determined. 

The soil remaining in the measuring flask was washed free 
from soluble nitrogen with a 1 per cent solution of potassium sul- 
fate by decantation from tall soil beakers, the solution after set- 
tling being syphoned ofif not oftener than twice a day. This meth- 
od was employed in order to prevent the clay from forming a 
colloidal suspension. Distilled water alone would remove all elec- 
trolytes and allow a portion of the clay to remain in the solution 
in colloidal suspension. It is known that suspensions of finely di- 
vided clay carry a negative charge in pure water. Since it is 
necessary for the complete precipitation of colloids to have some 
electrolyte present it Avas decided to use a 1 per cent solution of 
potassium sulfate. The negatively charged colloid was thus pre- 
cipitated by the positive ions of the potassium sulfate solution 
and at the same time the salt would not interfere with the subse- 
quent Kjeldahl determination. 

A concrete example of the thoroughness of this washing may 
well be given. It will be noted that 700 cc. of the original hydrol- 
ysate was syphoned off for the dififerent analyses. This left a total 
volume of 300 cc. of residue and solution to be washed by decanta- 
tion with 1 per cent potassium sulfate. By the methods of calcu- 
lation given in the following paragraphs it was found that the re- 
maining solution contained 0.1089 gram of nitrogen. If three- 
fourths of the wash solution is removed each time, there will remain 



in the solution at the end of the fourth washing approximately 0.0004 
gram of the original nitrogen. Actual Kjeldahl determinations 
were made on 250 cc. portions from the fourth washing in the case 
of duplicates from the same soil, and the results indicated that 
0.0006 gram of nitrogen still remained in the solution. Since this 
was within experimental error of the theoretical value, the method 
of washing by decantation was followed in all the subsequent work 
with mineral soils, or those which had mineral soils added previous 
to the analysis. 

- The residue from the h3'drolyzed soil was evaporated to dry- 
ness on the steam bath in an evaporating dish, then further dried 
at about 110° C. After cooling, this dry soil was passed through a 
1 mm. sieve and after being thoroughly sampled, duplicate nitro- 
gen determinations were made on 15 gram portions and the total 
nitrogen remaining in the soil calculated.- These results are listed 
as "insoluble humin nitrogen in the soil." The weight of the dry 
soil divided by the average specific gravity (2.6) represented the 
actual volume occupied by this soil residue. The total volume of 
the hydrolysate minus the volume occupied by the insoluble resi- 
due gives the actual volume of the soil solution. 

Since the analysis was made on 500 cc. of the soil solution it 
was necessary to recalculate the total "insoluble humin nitrogen in 
the soil" in order to determine the amount of this humin nitrogen 
actually belonging to the aliquot analyzed. 

The total nitrogen belonging to the solution analyzed was 
found by taking the sum of the total nitrogen in the solution and 
the above insoluble humin nitrogen. Knowing the total nitrogen 
content of the soil before hydrolysis and the total nitrogen in solu- 
tion, the per cent of the total nitrogen in solution after hydrolysis 
can be determined. 

The 500 cc. aliquot was concentrated under reduced pressure 
until the hydrochloric acid was practically removed and the am- 
monia nitrogen determined in the manner outlined under the peat 
analysis. 

The "humin" fraction precipitated by the calcium hydroxide 
was almost colorless or light yellow, due to the iron salts con- 
tained in it. This bulky precipitate was always washed by de- 
cantation after the method above described, except that distilled 
water was used, the united washings being concentrated to 200 
cc. or less for the precipitation of the basic nitrogen. 

It was found necessary to use 35 grams of phosphotungstic 
acid for the precipitation of the bases. The remainder of the 
analysis was carried out as directed under peats. 

3. The method for determination of "Jodidi numbers." A 
100 cc. portion of the clear hydrolysate was concentrated under 
reduced pressure and the ammonia nitrogen determined in the 
usual manner. The residue remaining in the flask after this de- 
termination was dissolved in concentrated hydrochloric acid and 
phosphotungstic acid added.* After standing the usual length of 
time the precipitate was filtered off and the total nitrogen deter- 

*It win~be noted that no "humin" fraction is separated. In that respect 
the "Jodidi numbers" differ from Hausmann numbers. 



38 

mined on the filter- and contents by the Kjeldahl method and listed 
as "basic N." The filtrate from the above precipitate was con- 
centrated and made to 300 cc. volume. Duplicate determinations 
were made on 100 cc. portions, and the total nitrogen listed as 
nitrogen in the filtrate from the "bases." 

4. The determination of nitrogen. Nitrogen was determined 
on the soils and soil extracts in the usual manner, using 25-35 cc. 
H.,S04, 10 gm. K.S( ).p and a crystal of CuSO.i. All titrations were 
made with N/14 acid and alkali so that the figures obtained repre- 
sented millierams of nitro<2"en without necessitating a calculation. 



D. The Analytical Data. 

The essential data which have already 1:»een published on the 
soils studied are shown in l'a1)le I. 

Tabic I . — Certain analylical data for the soils used iu this paper. 
Data of Gortjier (lpl6 a). 





1 


-b t; 




0) >. 








c/} ^ 


r^ 


J '?'> 


O 


C Ki 






C '3 






SI ^ 








fS 03 


o " 




i'i 


? >. 


^ 






cent 1 
n in 
sis). 


cen 
rbon 
t-y ba 


■A -^ m 


So 

03 


a 

_o 




S.S 


. CD nj 






53 .5 


"S 




fn 


Cu 


PW 


ft 


ft 


ft 


Calcareous black 














.§-rass-poat 


6.40 


2.940 


42.81 


0.600 


' 28.71 


14.56 


Sphagnum-covered 














peat 


5.90 


2 000 


49.32 


none 


32 91 


24.66 


Acid "'muck" soil. . . . 


5.60 


1.340 


14.58 


none 


7.14 


10.88 


Fargo clay loam .... 


3.92 


0.250 


2,678 


2.360 


2.66 


10.72 


Fargo silt loam 


14.89 


0.823 


10.02 


0.200 


9.91 


12.17 


Carrington silt loam. 


6,22 


0.371 


4.733 


0.090 


4.95 


12.76 


Hempstead silt loam 


3.07 


0.256 


2>.Zll 


0.020 


3.61 


13.17 


Prairie-covered loess 


7.89 


0.301 


3.704 


0.240 


3.40 


^ 12r30 


Forest-covered loess 


' 1.87 


0.128 


1.638 


0.120 


1.79 


12.79 



1. Analysis of "fibrin from blood" hydrolyzed in the presence 
of 100 grams ignited subsoil. 1'his analysis was conducted in order 
to ascertain if possible the eft'ect of soil minerals upon the hydro- 
lysis of a pure protein. Fibrin was selected because it was from 
a sample already analyzed ( Gortner 1916 c). The subsoil was first 
ignited to redness in a muffle furnace for an hour, in order to drive 
off all the organic matter, and subsequently cooled in a desiccator. 

Duplicate portions of five grams fibrin and 100 grams ignited 
subsoil were hydrolyzed in the presence of an excess of hydrochlo- 
ric acid. Upon the application of heat, fumes of hydrogen chloride 
were evolved for some time. The analysis was conducted like 
that of the mineral soils, excepting that a 600 cc. alic(Uot was used 
for the analysis, this amount of solution being equivalent to three 
grams of fibrin. After making- the ammonia determination the 
humin precipitate was washed by decantation in the usual man- 
ner and the filtrate made to 250 cc. volume. The first wash solu- 



39 



tion from the humin precipitate had a characteristic light blue 
color in each case. Duplicate Kjeldahl determinations were made 
on 25 cc. portions of this solution and the results listed as total- 
nitrogen-in-the-filtrate-from-humin. The remaining 200 cc. solu- 
tion was used for the precipitation of the bases and the subsequent 
analysis, but of course the results were calculated on the basis of 
the total volume. The total nitrogen belonging to the aliquot an- 
alyzed was determined by adding the nitrogen obtained as am- 
monia, humin, and total-nitrogen-in-the-filtrate-from-humin to the 
insolublc-humin-nitrogen-in-the-Roil. The filtrate-from-the-bases 
was made to 250 cc. volume. Twenty-five grams of phosphotungstic 
acid was used for the precipitation of the bases. The nitrogen 
retained by the barium phosphotungstate was 0.0022 gram for 
Sample I and 0.0020 gram for Sample II. 

The experimental data showing the grams of nitrogen found 
and per cent of total nitrogen are given in Table II. 

Tabic II. — Nitrogen distribution in three grains of Merck's "fibrin 
from blood" hydrolyzed in the presence of 100 grams of ignited subsoil. 



Grams nitrogen 



Per cent of total nitrogen 



I 



Total N I 0.4577 

Ammonia N • ■ | 0.0457 

Insoluble humin N in soil| 0.0125 
Humin N pptd. byl 

Ca(OH). ....[ 0.0220 

Basic N I 0.1219 

Argmme N • • | 0.0598 

Histidine N • ■ | none 

Lysine X • • I 0.0597 

Cystine N | 

Ajnino N in bases ........ j 

N in filtrate from bases..] 
Amino N in filtrate from] 

bases | 

Non-amino N in filtrate| 

from bases . . . j 0.0054 

Total N regained.......-! 0.4746 



II 



I 



II 



Av. 



0.4591 
0.0455 
0.0130 

0.0221 

0.0995 

0.0440 

none 

0.0531 



9.98 

2.73 

4.81 
26.63 
13.07 



9.91 

2.83 

4.81 

21.68 

9.58 



13.04 I 11.57 



0.0775 0.0746 
0.2725 i 0.2909 

0.2671 i 0.2702 

j 

0.0207 
0.4710 



16.93 
59.54 

58.36 

1.18 
103.69 



16.25 
63.36 

58.85 

4.51 
102.59 



9.95 

2.78 

4.81 

24.15 

11.32 

none 

12.30 

0.5^ 
16.59 
61.45 

58.61 

2.84 
103.14 



iProm data of Gortner (1916 c). 



Table III shows a comparison of these analyses with other 
analysis of the same sample of fibrin hydrolyzed alone, and in the 
presence of three times its weight of cellulose (data of Gortner 
1916 c). , 

Differences between these analyses, together with dififerences 
between duplicates in each series of analysis, and data showing 
"maximum" and "average" experimental differences to be expected 
are given in Table IV. 

These comparisons will be considered in detail under "Dis- 
cussion" in the latter part of this paper. 



40 



Table III. — Comparative analyses of three grams'^' of Merck's 
"fibrin from blood" hydrolysed alone and in the presence of carbo- 
hydrate and of ignited subsoil. 





rrl 


.-^ 


1 


1 


«M 


<(-( 




1 


1 




o 




o 


o 


«J 


o 


n 




o 


W 


-^ O 


t^ 


p 














>-l 






v 


a^ 


V 


p. 


n 





o 


-o 


P. 


W gm. 
subs 
data 


c 


a5 




c 


4) 


bu 


C3 


j3 
c 


0) 


j^ 


d 


C 


2 


C 


<4-l 

O 


0) 




X2 


c 


of 1 
ted 
age 
le II 


tC 


^ 


CC 






O 


tC 






Tl 



o 




-rt 




3 


•-<, 




■ri 


5) 




g 




0) 


O 

O 


s 

bJ3 




nee 
igni 

Aver 
Tab 


CO 






CO 










M 




0) -_> 



Per cent total iPer cent total I Per cent total 
nitrogen I nitrogen I nitrogen 



Ammonia N 

Humin N 

Arginine N 

Histidine N '...... 

Lysine N ■ • 

Cystine N 

Amino N in filtrate from bases . . . . 
Non-amino N in filtrate from bases 
Total N regained ••.... 



10.15 

2.83 
10.91 

4.36 
12.05 

0.51 
55.43 

2.51 
98.75 



9.85 
7.72 
8.56 
4.86 

11.04 
0.71 

52.02 
3.91 

98.67 



9.95 

7.59 

11.32 

none 

12.30 

0.51 

58.61 

2.84 

103.14 



*The three gram portion contained approximately 0.4550 gm. nitrogen. 

Table IV. — -Difference betzveen duplicate analyses (due to experi- 
mental errors) , the differences apparently dice to the addition of carbo- 
hydrate and of ignited subsoil, as zvell as Van Slyke's "maximum" and 
"average" differences to be expected. 



m 


u 




0) 


0) 










ai 








u 










^-0 




"C 


CM 




C 


o 




<D 






0) 


n1 












rf 




OJ 


.J 




^ 








a> 




c 






CD 


o 










0) 






iK 






-d 


fl 






u 




■*-' 


J2 


o 


OJ 


tC 




t 




1-( 




<H 




u 


O 




d 






Ph 







"SO 



C oi o 



^.HP 



^5 CO 

So"-" 



m 1 1 

CU.C 1 










o 






ts 


no) 1 






tj 


c 


c 


be 


<u 




<u<*-> 1 




O 














42 






be 


QJ 








c 


2 






S-" 


'O 


« 




■c 








.*- 




c 

Qj 








<D 


t'- 


Oh 





'C o 



nS. 



S fi^S 
ft >.+^ o 



£ '^ d 

s ^ p 



0) 



oo 

<U (DO 
bJ3 c [0 to 



Van Sb 


'ke's 


(1911") 


experimental 


differences 






c 




0) 




Cl 


c 




0) 




o 


CD 




& 


<D 


* 


P. 






C 


:: 




0) 


^ 


be 


c 


cri 


;^ 


t. 




^ 



Ammonia N. . . 

Humin N 

Arginine N . . • 
Histidine N. . . 



Lysine N.. . • • ■ 

Cystine N 

Amino N in fil 
trate from 
bases • ■ 

Non-amino N in 
filtrate from 
bases ..•-.. 



0.01 
0.11 
0.73 
0.07 

0.08 
0.10 



2.12 
0.27 



0.48 
0.25 
0.86 
0.66 

0.11 
0.00 



2.15 
0.40 



0.07 
0.10 
3.49 



1.47 



0.49 



3.23 



—0.30 
+4.89 
—2.35 
+0.50 

—1.01 
+0.20 



—3.41 



—0.20 
+4.76 
+0.41 
—4.36 

+0.25 



-3.18 



+ 1.40 +0.33 



0.37 
0.39 
1.27 
2.14 
(0.93)* 
1.23 
0.11 



1.60 
(0.60)^ 

1.20 



0.12 
0.20 
0.73 
0.79 

0.61 
0.05 



0.63 



0.68 



^The'ligure in parentheses rep 
tween duplicates observed by Van 



resents 
Slyke ( 



the second greatest 
1911). 



difference be- 



41 

2. Calcareous black grass-peat. Duplicate samples of 15 
grams were hydrolyzed for 48 hours in the presence of 100 cc. con- 
stant boiling hydrochloric acid, and the analysis conducted as de- 
scribed under the method for a peat soil. 

In the precipitation of the phosphotungstic acid with barium 
chloride, it was difficult to determine when the precipitation was 
complete. On addition of barium chloride a precipitate formed and 
the pink color slowly disappeared. Both sodium hydroxide and 
barium chloride were repeatedly added. The change in color oc- 
curred several times during a half-hour. At all times test portions 
gave immediate granular precipitates with both sodium sulfate 
and barium chloride. This indicated that we could not depend 
upon the test with sodium sulfate to determine when all the 
phosphotungstic acid was precipitated. After the addition of a 
large amount of barium chloride the solution was allowed to stand 
several hours in order to allow equilibrium to be established. At 
the end of this time test portions gave a heavy precipitate with 
sodium sulfate, but no action with barium chloride. In all subse- 
quent work, both with peat and mineral soils, the addition of barium 
chloride to the solution of the phosphotungstates was extended 
over an hour or more and the precipitate then allowed to settle 
over night. 

This barium phosphotungstate precipitate was washed thor- 
oughly by decantation with hot water until free of chlorides. This 
operation Avas carried out in the original beaker, and in the case of 
many mineral soils required from 500 to 1000 cc. to remove all 
chlorides. The washed precipitate of barium phosphotungstate and 
filter on kjeldahling gave 0.0021 gram nitrogen retained by Sample 
I and 0.0055 gram by Sample II. 

The experimental data showing the grams of nitrogen and 
per cent of tgtal nitrogen are given in Table V. 

Tabic v. — Nitrogen distribution in calcareous black grass-peat. 



Total N 

Ammonia N 

Humin N 

Basic N 

Basic N set free as NHs by 

50% KOH 

Basic N not set free as NH 

by 50% KOH...... 

Amino N of bases . . ■ • 

Non-amino N of bases , 

N in filtrate from bases 

Amino N in filtrate from 

bases 

Non-amino N in filtrate from 

bases • • • • 

Total N resrained ....-■.... 



Grams nitrogen 



I 



0.4412 
0.0833 
0.1145 
0.0494 

0.0150 

0.0344 
0.0309 
0.0185 
0.1932 

0.1810 

0.0122 
0.4404 



II 



0.4412 
0.0867 
0.1155 
0.0441 

0.0139 

0.0302 
0.0251 
0.0190 
0.1810 

0.1672 

0.0138 
0.4273 



Per cent of total nitr ogen 
A"^ 



I 



18.88 
25.95 
11.20 

3.40 

7.80 

7.00 

4.19 

43.79 

41.02 

2.77 
99.82 



II 



19.65 
26.18 
10.00 

3.15 

6.85 

5.69 

4.31 

41.02 

37.90 

3.12 
96.85 



19.26 
26.07 
10.60 

3.27 

7M 
6.35 

4,25 
42.40 

39.46 

2.94 
98.33 



42 



3. Sphagnum-covered peat. Duplicate 15 gram samples were 
hydrolyzed in the usual manner. It was found in the ammonia de- 
termination that all of the ammonia nitrogen could not be driven 
off in a half-hour when the volume of solution was large and a 
bulky precipitate of iron and aluminum hydroxides was present. 
Continued distillation for a further half-hour in this case gave 
additional ammonia nitrogen amounting to 0060 gram. In all sub- 
sequent work with both peats and soils the volume was kept smaller 
by the use of a more concentrated suspension of calcium hydroxide. 

The calcium hydroxide precipitate containing the "humin" 
nitrogen was difficult to digest in the subsequent Kjeldahl deter- 
mination, due to the large amount of carbonaceous organic material 
present. From 100-200 cc. of sulfuric acid were required for 
the digestion. After digestion the material was transferred to a 
1000 cc. flask and 250 cc. portions used for the distillation. It was 
often necessar}' to heat the flask on a sand bath to prevent severe 
bumping. This trouble was ol)viated in later work by adding zinc 
dust instead of granulated zinc to the distilling flask. The nitrogen 
retained by the barium phosphotungstate amounted to 0.0009 gram 
in Sample I. and 0.0013 gram in Sample II. 

The experimental data showing the grams of nitrogen and 
per cent of total nitrogen are g-iven in Table VI. 



Tabic VI. — Nitrogen distribution in spluu 



-Q'l'ercd peat. 



Grams nitrogen | Per cent of total nitrogen 



as 



NH: 



Total N . . 

Ammonia N 

Humin N 

Basic N 

Basic N set free 

by 50% KOH.......... 

Basic N not set free as 

NH3 by 50% KOH..... 

Amino N of bases 

Non-amino N of bases... 
N in filtrate from bases.. 
Amino N in filtrate from 

bases ■ • 

Non-amino N in filtrate 

from bases 

Total N regained 



I 

OJOOO 
0.0659 
0.0779 
0.0281 

0.0091 

0.0190 
0.0171 
0.0110 
0.1396 

0.1292 

0.0104 
0.3115 



II 
0.3000 
0.0737 
0.0804 
0.0303 

0.0088 

0.0215 
0.0145 
0.0158 
0.1184 

0.1135 

0.0049 
0.3028 



I 



21.97 

25.96 

9.37 

3.03 

6.32 

5.70 

3.67 

46.53 

43.06 

3.47 
103.83 



II 



Av. 



24.57 
26.80 
10.10 

2.93 

7.17 

4.83 

5.27 

39.46 

37.83 

1.63 
100.93 



23.27 

26.38 

9.73 

2.98 

6.75 

5.26 

4.47 

43.00 

40.45 

2.55 
102.38 



4. Acid "muck" soil One 25 gram sample waS' hydrolyzed 
in the presence, of 125 cc. concentrated hydrochloric acid for 48 
hours. The ammonia nitrogen was determined as usual, and the 
precipitate of calcium hydroxide* containing the "humin" was 
washed in a taW soil beaker in the manner described under the 
method for a mineral soil. After digestion of this precipitate, the 
material in the Kjeldahl flask was diluted to 500 cc. and duplicate 
distillations made on 250 cc. poi-tions. The bases were precipitated 
with 15 grams phosphotungstic acid. The total nitrogen retained by 
the barium phosphotungstate was 0.0022 gram. The solution of 
the frltrate-from-the-bases was made to a volume of 250 cc. 



43 



The experimental data showing the grams of nitrogen and per 
cent of total nitrogen are found in Table \TI. 

Table VII. — Nitrogen distribution in an acid "muck" soil. 



Grams 
nitrogen 



I'er cent 
of total 
nitrogen 



Total N . .^ • 

Ammonia N 

Humin N ■ • 

Basic N 

Basic N set free as NH^ by 50% KOH 

Basic N not set free as NH. by 50% KOH, 

Amino N of bases 

Non-amino N of liases 

N in filtrate from bases • • 

Amino N in filtrate from liases. . . . • • 

Non-amino N in filtrate from bases , 

Total N resained 



0.3350 
0.0653 
0.0925 
0.0454 
0.0104 
0.0350 
0.0306 
0.0148 
0.1300 
0.1133 
0.0167 
0.3332 



19.49 
27.61 
13.55 

3.10 
10.45 

9.13 

4.42 
38.81 
33.82 

4.99 
99.46 



5. Fargo clay loam. Duplicate portions of 250 grams were 
liydrol3'zed for 4S hours. The "huiain" precipitate recjuired 100 cc. 
sulfuric acid for the digestion. Alter digestion the material was 
transferred to a 500 cc. flask and 250 cc. portions used for the dis- 
tillation. This method was followed subsec|uently with the "humin" 
nitrogen determination of all the laineral soils. - 

The filtrate from "humin" was of a sirupy consistency in each ' 
case. The first addition of 15 grams of phosphotungstic acid did 
not entirely precipitate the bases. Five gram portions were added 
from time to time until a total of 50 grams had been used. After 
standing the usual length of time tfe precipitate of the bases was 
filtered ofi'. but even then the wash water caused the formation of 
a small additional precipitate in the filtrate. After warming on the 
steam bath this final solution was perfectly clear, and on standing 
oyer night, only a trace of precipitate was formed so the precipita- 
tion was considered complete. It appears probable that a portion 
of this precipitate is due to the formation of inorganic phospho- 
tungstates which consume a very large amount of the phospho- 
tungstic acid, for if all of this precipitate had consisted o^f basic 
nitrogen compounds the amount of nitrogen recovered should have 
beengreater than the amount which was actually found. In all 
the subsequent work 35 grams of phosphotungstic acid was used 
for the precipitation of the bases in the hydrolysates from mineral 
soils. 

The phosphotungstate precipitate dissolved very slowly in the 
sodium hydroxide as did all other phosphotungstic acid precipitates 
of the mineral soils studied. A Kjeldahl determination of the 
barium iihosphotungstate gave 0.0026 gram nitrogen. The solution 
containing the nitrogen of the bases was made up to 100 cc. volume. 

During the concentration of the filtrate from the bases so much 
precipitate separated, that this was filtered oft" and the solution 
made up to 300 cc. volume. The salt remaining was dissolved in 
water and also made up to a volume of 300 cc. Aliquot portions 
were taken from each solution and combined for the different de- 
terminations. 



44 



The experimental data giving the grams of nitrog-en and per 
cent of total nitrogen are found in Table V^III. 

Table VIII . — Nitrogen distribution in Fargo clay loam. 



Grams nitrogen 


Per cent 


of total nil 


rogen 




I 


II 


I ! 


II 


Av. 


Total N 


0.3368 


0.3338 








Ammonia N 


0.0788 


0.0821 


23.40 


24.60 


24.00 


Insoluble humin N in soil. 


0.0948 


0.0948 


28.15 


28.40 


28.27 


Humin N pptd. by 












Ca(OH). 


0.0352 


0.0266 


10.45 


7.97 


9.21 


Basic N 


1 


0.0320 




9.58 




Basic N set free as NHs 












by 50% KOH .. .. 




0.0108 




3.23 


3.23 


Basic N not set free as 












NHs by 50% KOH 




0.0212 




6.35 


6.35 


Amino N of bases. ....... 




0.0215 




6.44 




Non-amino N of bases. . • . 




0.0105 




3.14 




N in filtrate from bases. . . 




0.1092 




32.71 




Amino N in filtrate from 












bases 




0.1027 




30.77 


30.77 


Non-amino N in filtrate 




from bases 




0.0065 
0.3447 




1.95 
103.26 


1.95 


Total N regained , . . 


103.77 



1 Entire sample lost during precipitation of bases. 

6. Fargo silt loam. Two 125 gram portions were hydrolyzed 
•for 48 hours with 500 cc. hydrochloric acid (sp. gr. 1.18). During 
the first few hours large amounts of hydrogen chloride were 
evolved. 

The resulting hydrolysates from the two flasks were com- 
bined and diluted to 2 liters. After settling, a 1 liter portion was 
syphoned off and analyzed by the usual method. Two 100 cc. por- 
tions M'-ere used for the determination of total nitrogen in solu- 
tion, and another portion of 200 cc. was used for determination 
of the "Jodidi numbers." The nitrogen retained by the barium 
phosphotungstate was 0.0091 gram. The solution containing the 
basic nitrogen was made to 100 cc. volume and that containing 
the nitrogen in the' filtrate from the bases to 300 cc. volume. 

The experimental data showing grams of nitrogen and per 
cent of total nitrogen are given in Table IX. 

Table IX. — Nitrogen distribution in Fargo silt loam. 



Grams 
nitrogen 



Per cent 
of total 
nitrogen 



Total N . . 

Ammonia N • 

Insoluble humin N in soil 

Humin N pptd. by CaCOH). 

Basic N • • • 

Basic N set free as NH. by 50% KOH..., 
Basic N not set free as NH;, l)y 50% KOH, 

Amino N of bases • ■ • 

Non-amino N of bases • , 

N in filtrate from bases 

Amino N in filtrate from bases 

Non-amino N in filtrate from bases , 

Total N regained 



0.9238 
0.2454 
0.2118 
0.030r 
0.1119 
0.0296 
0.0823 
0.0695 
0.0424 
0.3246 
0.3015 
0.0231 



26.56 

22.93 

3.26 

12.11 

3.20 

8.91 ' 

7.52 

4.59 

35.14 

32.64 

2.50 

100.00= 



'■ By difference. 



•' Calculated. 



45 



7. Carrington silt loam. Sample I (Nerstrand orig-in). The 
hydrolysis of 250 grams was carried out according to the method 
described under Fargo silt loam. The "humin" nitrogen was de- 
termined as outlined under Fargo clay loam. Sample II (Morris- 
town origin) was hydrolyzed and the same methods of analysis 
employed throughout as was applied to Sample I. The nitrogen 
retained by the barium phosphotungstate precipitate was 0.0028 
gram in Sample I and 0.0025 gram in Sample II. In both samples 
the solutions containing the bases and filtrate from the bases were 
made to the same dilution as under Fargo silt loam. 

The experimental data showing grams of nitrogen and per cent 
of total nitrogen are given in Table X. 

Table X. — Nitrogen distribution in Carrington. silt loam. 



Sample No. I 



Sample No. II 



Grams 
nitrogen 



Per cent 
of total 
nitrogen 



Grams 
nitrogen 



Per cent 
of total 
nitrogen 



Total N 

Ammonia N 

Insoluble humin N in soil 

Humin N pptd. by Ca(OH).. 

Basic N • • . . 

Basic N set free as NHs by 50% 

KOH 

Basic N not set free as NH?, by 50% 

KOH 

Amino N of bases 

Non-amino N of bases 

N in filtrate from bases 

Amino N in filtrate from bases 

Non-amino N in filtrate from bases. 
Total N reR'ained . • 



0.5124 
0.1463 
0.1324 
0.0304 
0.0600 

0.0172 

0.0428 
0.0412 
-0.0'188 
0.1305 



0.4996 



28.55 

25.84 

5.93 

11.71 

3.36 

8.35 
8.04 
3.67 

25.47 



97.50 



0.4920 

0.1381 

0.1210 

0.0326' 

0.0725 

0.0156 

0.0569 
0.0393 
0.0332 
0.1390 
0.1272 
0.0118 
0.5032 



28.07 

24.59 

6.63 

14.74 

3.17 

11.57 

7.99 

6.75 

28.25 

25.85 

2.40 

102.28 



^Solution lost at this point. 

' Result calculated from the dig-estion of one-half the precipitate. 

8. Hempstead silt loam. Duplicate 250 gram samples were 
hydrolyzed for 48 hours. The "humin" was washed and the de- 
termination carried out as described under mineral .soils. The only 
explanation which occurs for the higher ammonia nitrogen of one 
hydrolysate is that Sample I must have been heated inore strongly 
during hydrolysis. From unpublished experiments conducted at 
this Station it has been found that when a pure protein is hydro- 
lyzed under vigorous boiling, a greater amount of ammonia nitro- 
gen is invariably obtained than when hydrolyzed under slow boil- 
ing conditions. This observation was made subsequently to my 
own, so that the importance of the rate of boiling was not known 
at the time of doing my work. 

Only 25 grams of phosphotungstic acid were added for the 
precipitation of the bases. After standing the solution gave a 
further precipitate on addition of more phosphotungstic acid. It 
Avas concluded, however, that the organic bases were entirely pre- 
cipitated, for on washing the precipitate with the phosphotungstic 
acid wash water no additional precipitate formed. The nitrogen 
determination gave 0.0011 gram retained by the barium phospho- 
tungstate. The solution containing the filtrate from the bases was 
made to 300 cc. volume. 



46 



The experimental data giving the grams of nitrogen and pel 
cent of total nitrogen will be found in Table XI. 

Table XI. — Nitrogen distribution in Hempstead silt loam. 



Gr ams nitrogen 

~i 1 IT' 



Per cent of total nitrogen 



I 



II 



Av. 



0.0164 
0.0389 

0.0078 

0.0311 
0.0254 
0.0135 
0.0960 

0.0839 



30.50 
28.72 

4.80 
11.39 

2.28 

9.10 

7.44 

3.95 

28.10 

24.56 

3.54 
103.51 




29.44 

27.56 

4.80 



2.28 
9.10 

24.56 

3.54 
101.29 



Total N I 0.3416 | 0.3485 

Ammonia N | 0.1042 | 0.0989 

Insoluble humin N in soilj 0.0981 | 0.0920 
Humin N pptd. b}' 

Ca(OH). 

Basic N 

Basic N set free as NHs 

by 50% KOH .. 

Basic N not set free as 

NH. by 50% KOH 

Amino N of bases 

Non-amino N of bases. . • • 
N in filtrate from bases. . . 
Amino N in filtrate from 

bases 

Non-amino N in filtrate] 

from bases | 0.0121 

Total N regained_. . |_ 0.3536 

^Solution lost at this point. 

9. Prairie-covered loess. In Sample I, 250 grams were hy- 
drolyzed in the presence of 250 cc. of constant boiling hydrochloric 
acid for 48 hours. The hydrolysate on cooling was diluted to 1000 
cc.and a 500 cc. portion was syphoned off and used for the an- 
alysis. In Sample II, two 125 gram samples were hydrolyzed in 
the same manner as outlined under Fargo silt loam (500 cc. con- 
stant boiling hydrochloric acid to 125 grams soil). The dilution 
and aliquot tised for analysis were also the same. The nitrogen 
retained by the barium phosphotungstate amounted to 0.0020 gram 
in Sample I, and 0.0072 gram in Sample II. The solution con- 
taining the bases w^as diluted to 100 cc. in both samples. 

The experimental data showing grams of nitrogen found and 
per cent of total nitrogen are gi^'en in Table XII. 

Table XJI. — Nitrogen distribution in prairic-covcred loess. 





Grams nitrogen 


Per cent 


of total nitrogen 




I 

0.3907 


II 


i 1 II 


Av. 


Total N 


0.4012 








Ammonia N . 


0.1223 


0.1194 


31.30 


29.76 


30.53 


Insoluble humin N in soil 


0.0922 


0.1007 


23.60 


25.10 


24.35 


Humin N pptd. by 












Ca(OH). 


0.0198 


0.0213 


5.07 


5.31 


5.19 


Basic N 


0.0444 


0.0562 


11.36 


14.01 


12.68 


Basic N set free as NHs 












by 50% KOH 


0.0100 


0.0132 


2.56 


3.29 . 


2.92 


Basic N not set free as 












NH:, by 50% KOH 


0.0344 


0.0430 


8.80 


10.72 


9.76 


Amino N of bases 


0.0271 


0.0322 


6.93 


8.03 


7.48 


Non-amino N of bases... 


0.0173 


0.0240 


4.43 


5.98 


5.20 


N in filtrate from bases.. 


0.1152 


0.1128 


29.49 


28.12 


28.80 


Amino N in filtrate from 












bases 


0.1044 


0.1033 


26.72 


25.75 


26.24 


Non-amino N in filtrate 




from bases . . 


0.0108 
0.3939 


0.0095 
0.4104 


2.76 
100.82 


2.37 
102.30 


2.56 


Total N regained 


101.56 



47 



10. Forest-covered loess. In Sample 1, 300 grams of soil 
were hydrolyzed and diluted in the same manner as Sample I of 
prairie-covered loess. In Sample II, 300 grams of soil were hy- 
drolyzed under the same conditions as Sample II of prairie-covered 
loess. The nitrogen retained by the barium phosphotungstate was 
0.0024 gram in Sample I, and 0.0023 gram in Sample II. The 
solution containing the nitrogen of the bases was diluted to 100 
cc. in both samples and the solutions containing the total nitrogen 
of the filtrates were made to a vohrnie of 300 cc. 

It is observed that the volume of acid used in the hydrolysis 
had little effect on the proportion of the different fractions. The 
only observed difference is in the insoluble humin nitrogen retained 
by the soil residue, and this is slightly larger in Sample II, which 
was hydrolyzed in the presence of the greatest excess of acid. In 
connection with this it must also be noted that there is a somewhat 
larger quantity of nitrogen in solution in Sample II than in Sample 
I. Much the same results are shown with the prairie-covered loess. 
All increases or decreases in the various fractions due to the greater 
excess of acid may well be considered to be within the experimental 
error. 

The experimental data showing grams of nitrog-en found and 
per cent of total nitrogen are given in Table XIII. 

Table XIII. — Nitrogen distribution in forest-covered loess. 



Total N 


0.2224 
0.0646 
0.0574 


Ammonia N 


Insoluble humin N in soil 


Humin N pptd. by 




Ca(OH).> 


0.0140 1 


Basic N 


0.0316 


Basic N set free as NHs 


■ by 50% KOH 


0.00/4 


Basic N not set free as 




NH:, by 50% KOH 


0.0242 


Amino N of leases 


0i.O175 


Non-amino N of bases. . . . 


0.0141 


N in filtrate from bases.. 


0.0621 


Amino N in filtrate from 




bases • ■ . 


0.0585 


Non-amino N m filtrate 




from bases 


0.0036 
0.2297 


Total N regained 



Grams nitrogen__ 
~I \ II~ 

"0'2362" 
0.0669 
0.0662 



0.0080' 
0.0325 

0.0096 

0.0229 
0.0172 
0.0153 
0.0626 

0.0568 

0.0058 



Per cent of total nitrogen_ 
I 



^ By difference. 



Calculated. 




11. Sphagnum-covered peat hydrolyzed in the presence of nine 
times its weight of a mineral subsoil. Duplicate 10 gram samples 
were hydrolyzed in the presence of 90 grams subsoil with constant 
boiling hydrochloric acid for 48 hours. The hydrolysate was con- 
centrated as mucii as possible and ammonia nitrogen determined 
on the entire mixture by distillation with an excess of calcium hy- 
droxide for one hour.* 

*This was the first attempt to determine the nitrogen fractions in the 
presence of a mineral soil. For certain reasons later analyses have already 
been reported in this paper. The analyses as reported in this paper are 
by no means in chronological order, which may explain seeming inconsist- 
encies. 



48 

The residue remaining after hydrolysis was washed free of 
chlorides on ordinary funnels. It was then digested in three 
Kjeldahl flasks using a total of 30 grams potassium sulfate and 
360 cc. sulfuric acid. After digestion the material was transferred to 
a 1000 cc. flask, made to volume, and the nitrogen content deter- 
mined as described under sphagnum-covered peat. It will be ob- 
served that the total "humin" nitrogen is determined here instead 
of being reported in two separate fractions as was done in the 
case of all other analyses containing mineral soil. 

The combined filtrate and washings from the "humin" precipi- 
tate were concentrated in the usual manner and made to 200 cc. 
volume. Duplicate Kjeldahl determinations were made on 25 cc. 
portions of this solution and the results listed, as total nitrogen- 
in-the-filtrate-from-humin. The remaining 150 cc. portion was 
used for the precipitation of the bases, the subsequent procedure 
being completed as described in. the "fibrin from blood'' analysis. 
The barium phosphotungstate precipitate retained 0.0016 gram of 
nitrogen in Sample I, and 0.0053 gram in Sample II. 

In the second sample the combined filtrate and washings 
from the phosphotungstic acid precipitate of the bases were brought 
to near the neutral point with 50 per cent sodium hydroxide and a 
small amount of acetic acid added at once. After the addition of 
the acid it seemed possible that the neutral point had not been 
reached the first time, so more sodium hydroxide was added until 
the neutral point was just passed and acetic acid again added. The 
resulting solution was placed in a double-necked distilling flask 
and an attempt made to concentrate the solution under diminished 
pressure. Frothing was so intense that it was impossible to effect 
any concentration. When alcohol was added the^ distillation con- 
tinued quietly as long as any alcohol was present, but after that the 
frothing continued. The mixture behaved like a concentrated soap 
solution. The solution was finally concentrated in an evaporating 
dish over a water-bath and the resulting solution made to 300 cc. 
volume. On shaking the solution frothed very badly. 

The experimental data showing the grams of nitrogen found 
and the per cent of total nitrogen are given in Table XIV. 

A comparison between these analyses and those of the peat 
hydrolyzed alone is made in Table XV, the data of the peat hy- 
drolyzed alone being taken from Table VI, and recalculated from 
a 15 gram basis to a 10 gram basis. 



49 



Table XI J \ — Nitrogen distrihution i 
hydrolysed in the presence of nine times its ■ 



n s phagnum-covered peat 
weight of a mineral subsoil. 



of to tal nitrogen 

: II i~~at: 




Total N 

Ammonia N 

Humin N 

Basic N . • 

Basic N set free a 

by 50% KOH 

Basic N not set free 

NH. by 50% KOH 

Amino N of bases • • 

Non-amino N of bases. . . . 
N in filtrate from bases.. 
Amino N in filtrate from 

bases • .. . 

Non-amino N in filtrate 

from bases 

Total N re.sained 



0.0118 
0.2505 



' These results are evidently incor 
figures from the first column only are 



erage" column the 



Table XV. — Comparative analyses of sphagnum-covered peat 
hydrolysed alone and in the presence of nine times its iveight of a min- 
eral subsoil. 



Grams Nitrogen 



Peat 



Peat '^Increase (4 ) 
+ \i or 

Subsoil' / Decrease( — ) 



Apparent 
distribution 

of N in 

subsoil in 

per cent of 

total 

nitrogen 



Total N 

Ammonia N 

Humin N 

Basic N 

Basic N set free as NHs by 

50% KOH 

Basic N not set free as NH3 

by 50% KOH 

Amino N of bases . 

Non-amino N of bases ...... 

N in filtrate from bases.... 

Amjno N in filtrate from 

bases 

Non-amino N in filtrate from 

bases 

Total N regained .......... 



0.2000 
0.0466 
0.0527 
0.0195 

0.0060 

0.0135 
0.0105 
0.0090 
0.0860 

0.0776 

0.0084 
0.2048 



0.2441 
0.0623 
0.0658 
0.0268 

0.0100 

0.0168 
0.0178 
0.0090 
0.0951 

0.0825 

0.0126 
0.2500 



+0.0441 
-f0.01S7 
+0.0131 
+0.0073 

+0.0040 

+0.0033 
+0.0073 



-0.0091 

-0.0049 

-0.0042 
-0.0452 



35.60 
29.71 
16.55 

9.07 



16.55 

' 20.64 

11.11 

9.52 
102.50 



^ Ninety g-m. of subsoil contained 0.0441 gm. of soil nitrogen. 

12. Sphagnum-covered peat hydrolyzed in the presence of 
metallic tin. This peat was hydrolyzed in the presence of a reduc- 
\ng agent because it was thought that the amount of "humin" nit- 
rogen would be reduced, for according to Samuely (1902) the 
formation of this dark colored product is due to an oxidative proc- 
ess. Hlasiwetz and Habermann (1871 and 1873) hydrolyzed pro- 
tein with hydrochloric acid in the presence of stannous chloride in 
order that the solution should remain colorless. Cohn (1896-97 



50 



and 1898-99) believed that the use of a reducing agent was not 
essential, but according to Otori (1904) this is a mistake. 

It is perhaps significant that the "humin" nitrogen was reduced 
to 3.90 per cent by the presence of a reducing solution. It is not 
known whether there was sufficient tin present to maintain a reduc- 
ing solution throughout the hydrolysis inasmuch as the ferric iron 
in the peat would have an oxidizing action on the stannous salt. 
The sample was known to contain iron but the amount was not 
d( termined. 

Duplicate 15 gram samples were hydrolyzed with 100 cc. hydro- 
chloric acid (sp. gr. 1.115) for 48 hours in the presence of five and 
ten grams of tin respectively. The tin was first partially dissolved 
in the acid before the samples of peat were added. The deter- 
mination of "humin" nitrogen was carried out as directed under 
sphagnum-covered peat excepting that the digested material was 
diluted to 500 cc. instead of to 1 liter. The nitrogen retained by 
the barium phosphotungstate was 0.0023 gram in Sample I, and 
0.0047 gram in ^Sample II. The solution containing the filtrate from 
the bases was diluted to a volume of 300 cc. The experimental data 
giving the grams of nitrogen found and the per cent of total 
nitrogen are given in Table XVI. 

Table XVI. — Nitrogen distribution in sphagnum-covered peat 
hydrolyzed in the presence of metallic tin. 



Grams nitrogen 



Per cent of total nitrogen 





1 I 


II 


I 1 


II 


Av. 




5 gm. tin 

"OJOOO" 

0.0633 


10 gm. tin 

"0.3000 " 
0.0578 








Total N 


"21". i 6 


' V9.27 




Ammonia N • • . . 


20.18 


Humin N 


0.0699 
0.0320 


0.0650 
0.0417 


23.30 
10.67 


21.67 
13.90 


22.48 


Basic N 


12.28 


Basic N set free as NHs 




by 50% KOH •• 


0.0099 


0.0100 


3.30 


i.ii 


3,31 


Basic N not set free as 












NH, by 50% KOH.... 


0.0221 


0.0317 


7.Z7 


10.57 


8.97 


Amino N of bases ..••.,.■■ 


0.0193 


0.0219 


6.43 


7.30 


6.86 


Non-amino N'of bases. . . . 


0.0127 


0.0198 


4.23 


6.60 


5.41 


N in filtrate from bases.. 


0.1404^ 


0.1380 


46.80 


46.00 


46.40 


Amino N in filtrate from 












bases 


0.1297 


0.1307 


43.23 


43.57 


43.40 


Non-amino N in filtrate 






0.0107 
0.3056 


0.0073 
0.3025 


3.56 
101.86 


2.43 
100.83 


2.99 


Total N regained 


101.34 



1 This result is from a single determination of nitrog-en. 

13. Analysis of a 1 per cent hydrochloric acid extract of sphag- 
num-covered peat and (in part) of calcareous black grass-peat. 

Acid extraction was made of the two peats in direct contact with 
1 per cent hydrochloric acid. For the extraction 125 gram por- 
tions were placed in 2.5 liter acid bottles and two liters of 1 per 
cent acid added. In the case of calcareous black grass-peat, how- 
ever, the calculated amount of hydrochloric acid necessary to neu- 
tralize the calcium oxide was first added and then sufficient dilute 
acid to make two liters of a 1 per cent solution. Five hundred 



51 

grams were taken in the case of sphagnum-covered peat, while 750 
grams were taken in the case of calcareous black grass-peat. The 
bottles were shaken at intervals during five days and then the con- 
tents filtered through two thicknesses of a good grade oi cheese 
cloth and squeezed in the hands. The resulting solution was then 
filtered through two thicknesses of filter paper on a Buchner funnel. 

This extract was colored in each case but the calcareous black 
grass-peat gave a deeper straw colored solution than did the sphag- 
num-covered peat. This was probably due to tlie presence of a 
larger amount of iron in the one case than in the other. The cal- 
careous black grass-peat is known to contain a very considerable 
quantity of iron. The wash water in both instances was also straw 
colored. 

It has been shown l)y a number of investigators, e. g., Jodidi 
(1909), Kclley and Thompson (1914), and Gortner (1916 a) that 
considerable amounts of nitrogen are dissolved from certain soils 
by this preliminary treatment. The acid solution thus obtained 
should contain the ammonia, acid amides, amines, amino acids, and 
all other organic nitrogenous compounds soluble in water or very 
dilute acid. The 1 per cent hydrochloric acid extracted 8.57 per cent 
of the total nitrogen from sphagnum-covered peat, and 5.09 per 
cent from the calcareous black grass-peat. 

Duplicate nitrogen determinations were made on 250 cc. por- 
tions of the acid extract and from these results the total nitrogen 
in the bulk solution determined. The 5500 cc. solution from sphag- 
num-covered peat, containing 0.6468 gram nitrogen, and the 5000 
cc. from the calcareous black grass-peat, containing 0.4690 gram 
nitrogen, were used for analysis. These solutions were concen- 
trated under reduced pressure to about 200 cc. and then hydrolyzed 
for 48 hours, after first adding 75 cc. concentrated hydrochloric 
acid to the solution from sphagnum-covered peat, and 100 cc. to 
the solution from the calcareous black grass-peat. During evap- 
oration under reduced pressure considerable hydrolysis took place 
for the solutions turned dark brown in color. During hydrolysis 
of calcareous black grass-peat silicic acid separated in the con- 
denser.* 

The analysis of sphagnum-covered peat shows that over 65 per 
cent of the nitrogen is in the form of ammonia. Potter and Snyder 
(1915 a) have shown that a very small amount of the nitrogen 
in the 1 per cent hydrochloric acid extract of soils exists in the soil 
as ammonia nitrogen. It seemed probable that if an extract of the 
peat contained so much ammonia nitrogen after hydrolysis, the air 
dry peat must contain an appreciable amount in the ordinary con- 
dition. The ammonia nitrogen was determined directly on a 5' gram 
sample of the air dry material. An excess of calcium hydroxide 
solution was added and the mixture distilled under reduced pressure 
for forty-five minutes. It was found that 5.40 per cent of the total ■ 



*This was also true with all of the mineral soils studied, and is probably 
due to the presence of inorganic fluorides. 



52 

nitrogen of the soil existed in the form of ammonia nitrogen.* 

The precipitates containing the "humin" nitrogen were 
washed by decantation until practically all the dissolved nitrogen 
was removed. After digestion the material was diluted to 500 cc. 
and 250 cc. portions used for distillation. Before concentration the 
filtrate from the "humin" precipitate of sphagnum-covered peat 
was reddish in color and after concentration this color changed to a 
cherry red. Twenty-five grams of phosphotungstic acid was used 
for precipitation of the bases in sphagnum-covered peat. The 
barium phosphotungstate precipitate retained 0.0022 gram nitrogen. 
The solution containing the basic nitrogen was diluted to 50 cc. 
and the one containing total nitrogen-in-filtrate-from-bases to 250 
cc. 

The experimental data showing the grams of nitrogen found 
and per cent of total nitrogen are given in Table XVII. 

Tabic XVII. — Nitrogen distribution of a 1 per cent hydrocliloric 
acid extract of sphagmim-covcrcd peat and {in part) of calcareous 
black grass- peat. 



Sphagnum-covered 
peat 



Grams 
nitrogen 



Per cent | f^j.^^c, 
of total }^,l^'^l, 
r,u,-oo-oT, nitrogen 



Calcareous 
black grass-peat 
Per cent 
of total 
nitrogen 



Total N 

Ammonia N 

Humin N 

Basic N 

Basic N set free as NH3 by 50% 

KOH 

Basic N not set free as NH-, by 50% 

KOH 

Amino N of bases 

Non-amino N of bases 

N in fihrate from bases 

Amino N in filtrate from bases 

Non-amino N in filtrate from bases. 
Total N regained 



0.6468 
0.4230 
0.0657 
0.0389 

0.0181 

0.0208 
0.0266 
0.0123 
0.1335 
0.1107 
0.0228 
0.6611 



65.40 

10.16 

6.01 

2.80 

3.21 

4.11 

1.90 

20.64 

17.11 

3.53 

102.21 



0.4680 
0.3013 
0.0582 



64.38 
12.44 



' Distribution of remaining nitrogen not determined. 



14. Analysis of a portion of sphagnum-covered peat soluble 
in 4 per cent sodium hydroxide and precipitated by hydrochloric 
acid and (in part) of a similar solution from a calcareous black 
grass-peat. The- organic m.aterial soluble in 4 per cent sodium 
hydroxide was next extracted from new portions of the two differ- 
ent peats. Twelve 5 gram portions were leached with 1 per cent 
of hydrochloric acid to the absence of calcium and the excess of 
acid removed by washing with distilled water, until the filtrate 
indicated only a faint trace of free acid when tested with Squibb's 
litmus paper. After leaching and washing, each 5 gram portion 
was washed into tall glass-stoppered cylinders of 500 cc. capacity 



*This was further indicated by g-reenhouse experiments. The peat was 
treated with calcium carbonate at the rate of 4000 pounds per acre and planted 
to barley. The plants made a very rapid growth during the early stag'es of 
development and finally lodged. Next to this was a plot of calcareous black 
grass-peat which contained only 0.88 per cent of its total nitrogen in the 
form of ammonia nitrogen. When limed and sown to barley, it did not show 
any abnormal growth. 



53 

with 4 per cent sodium hydroxide and filled to the mark. These 
were thoroughly shaken and placed on their sides, thus allowing 
the peat to settle on the sides of the cylinder, thereby exposing a 
very large surface to the action of the hydroxide. The shaking was 
repeated at intervals for nine days. The cylinders were then thor- 
oughly shaken, placed in a vertical position and allowed to settle 
for four days before the supernatant liquid was syphoned off and 
filtered. The samples of calcareous black grass-peat were almost 
completely dissolved by the hydroxide solution. 

These filtered solutions were neutralized with hydrochloric 
acid (solution tested faintly acid) when a brown flocculent precipi- 
tate separated. This was allowed to settle for several hours and 
the cider colored solution syphoned off. The brownish black pre- 
cipitates were filtered and after draining over night were thoroughly 
mixed with a large volume of water and again filtered and drained. 
The resulting precipitates were hydrolyzed with 200 cc. of hydro- 
chloric acid for 48 hours. This amount of concentrated acid was 
added and the flask brought to boiling until hydrogen chloride 
fumes were evolved showing the presence of constant l)oiling acid. 
Silicic acid separated on the condenser during the hydrolysis of 
calcareous black grass-peat. Even after the hydrolysis there still 
remained some small lumps of the "humus" precipitate. 

The entire hydrolysate was used for the ammonia determina- 
tion. After this determination the "humin" precipitate was thor- 
oughly ground in a mortar to insure complete disinteg-ration, al- 
though this seemed hardly necessary as the solid was already in 
a fairly fine state of division. This precipitate was washed in the 
usual manner by decantation, the filtrate concentrated by evapora- 
tion and made to 250 cc. volume. Duplicate portions of 25 cc. 
were used for the determination of total nitrogen in the solution 
and the result listed as total nitrogen-in-the-filtrate-from-humin. 
The remaining 200 cc. portion was used for precipitation of the 
bases and subsequent analysis. 

The high content of carbonaceous organic matter made the 
"humin" precipitate xevy difficult to digest. The sulfuric acid 
required-was 120 cc. and the dig'estion extended over 10 days be- 
fore complete decoloration was effected. Of course precautions 
were taken to prevent the absorption of ammonia from outside 
sources. The material was diluted to 500 cc. and 250 cc. portions 
used in the distillation. The basic nitrogen was precipitated with 
25 grams of phosphotungstic acid. The nitrogen retained by the 
barium phosphotungstate was 0.0043 gram. The solution contain- 
ing the basic nitrogen was made to 50 cc. volume, and that con- 
taining thr total nitrogen-in-the-filtrate-from-the-bases was made 
to a volume of 2.50 cc. 

The experimental data showing the grams of nitrogen and per 
cent of total nitrogen are given in Table XVIII. 



54 



Table XVIII. — Nitrogen distribution in that portion of a sphag- 
num-covered peat soluble in 4 per cent sodium hydroxide and precipi- 
tated by hydrochloric acid and (in part) of a similar solution from a 
calcareous black grass-peat. 



1 


Sphagnum-covered 1 
peat 1 


Calcareous 
black grass-peat 




Grams 
nitrogen 


Per cent 
of total 
nitrogen 



16.22 
33.22 
10.80 

2.45 

8.35 

6.08 

4.72 

41.08 

33.74 

7.34 

101.32 


Grams 
nitrogen 


Per cent 
of total 
nitrogen 


Total N 

Ammonia N 


0.2860 
0.0464 
0.0950 
0.0309 

0.0070 

0.0239 
0.0174 
0.0135 
0.1175 
0.0965 
0.0210 
0.2898 


0.6344 
0.0781 

0.2093 

1 


"12.31 


Humin. N 


32.99 


Basic N 

Basic N set free as NHs by 50% 
KOH 




Basic N not set free as NHs by 50% 
KOH 




Amino N of bases 




Non-aniino N of bases 




N in filtrate from bases 




Amino N in filtrate from bases 

Non-amino N in filtrate from bases. 
Total N regained 





Distribution of the remaining nitrogen not determined. 

15. Analysis of a portion of sphagnum-covered peat soluble 
in 4 per cent sodium hydroxide and not precipitated by hydro- 
chloric acid and (in part) of a similar solution from a calcareous 
black grass-peat. The filtrate remaining from the brownish black 
precipitate formed by acidifying the sodium hydroxide extracts 
of the soil with hydrochloric acid (cf. section 13) were concentrated 
in the usual manner to about 700 cc. when a heavy precipitate of 
sodium chloride separated. On standing over night there also 
separated a heavy flocculent brown precipitate. This may have 
been due to the salting out effect of the sodium chloride on some 
of the organic substances in the solution. The solution was sat- 
urated with hydrogen chloride in the cold and the mixture then 
divided into two portions and hydrolyzed for 48 hours. The cold 
material after hydrolysis was united and filtered through glass 
wool and the precipitate washed with concentrated hydrochloric 
acid. The filtrate was allowed to stand in a tall soil beaker when 
more salt separated, due to the increased concentration of the hydro- 
chloric acid. The salt that separated was freed from the mother 
liquid by packing in a centrifuge and washing with acid a number 
of times. The salt washed as free of the solution as possible was 
dried on the steam bath. It was nearly white in color. The glass 
wool was dried and ground with the salt. After being sampled, 15 
gram portions were used for Kjeldahl determinations. The results 
were listed as nitrogen-retained-by-the-salt. 

The combined filtrates were concentrated and analyzed. After 
the digestion of the humin nitrogen, the material in the Kjeldahl 
flask was made to 500 cc. volume and the distillations carried out 
on 250 cc. portions. The filtrate and washings from the humin 
were acidified and evaporated under diminished pressure to less 
than 200 cc. and then made to 250 cc. volume. Duplicate nitrogen 
determinations were made on 25 cc. portions of the solution and 



55 



the results listed as total nitrogen-in-the-filtrate-from-humin. 

The total nitrog-en in the hydrolysate was determined by add- 
ing the nitrogen obtained as ammonia, and humin (in salt and that 
precipitated by calcium hydroxide) to the total nitrogen-in-the- 
filtrate-from-humin. 

Fifteen grams of phosphotungstic acid were used for the pre- 
cipitation of the base's. The barium phosphotungstate from the 
sphagnum-covered peat retained 0.0055 gram nitrogen. The solu- 
tion containing the basic nitrogen was made to^ 50 cc. \"^in-i- -- 
that containing the total nitrogen-in-the-filtrate-from-the-bases was 
made to 250 cc. volume. 

The experimental data showing the grams of nitrogen and per 
cent of total nitrogen are given in Table XIX. 

Table XIX. — Nitrogen distrihntion in that portion of a sphagnum- 
covered peat soluble in 4 per cent sodium hydroxide and not precipi- 
tated by hydrochloric acid and (in part) of a similar solution from a 
calcareous black grass-peat. 



Sphagnum-coveied 
peat 



Calcareous 
black grass-peat 



Total N 

Ammonia N 

Humin N pptd. by CaCOH)^ 

Humin N retained in NaCl 

Basic N 

Basic N set free as NHs by 50% 

KOH 

Basic N not set free as NHs by 50% 

KOH 

Amino N ^f bases 

Non-amino N of bases 

N in filtrate from bases 

Amino N in filtrate from bases 

Non-amino N in filtrate from bases. . 
Total N regained 



Grams 
nitrogen 

~ 0.3736 
0.0993 
0.0335 
0.0102 
0.0324 

0.0070 

0.0254 
0.0160 
0.0164 
0.2006' 
0.1721 
0.0285 
0.3760 



Per cent 
of total 
nitrogen 



26.58 
8.97 
2.73 
8.67 

1.87 

6.80 

4.28 

4.39 

53.69 

46.07 

7.62 

100.64 



Grams 
nitrogen 



[Per cent 

of total 

I nitrogen 



0.6204 
0.2003 
0.0652 
0.0169 



32.29 
10.51 

2.72 



* Calculated from one determination. Duplicate lost in digestion. 
2 Distribution of the remaining nitrogen not determined. 

16. "Jodidi numbers." These determinations were carried 
out as directed previously on Fargo clay loam, Fargo silt loam, 
Hempstead silt loam, prairie-covered loess, and forest-covered 
loess. 

The resulting data in grams and in per cent of total nitrogen 
are shown in Tables XX, XXI, XXII, XXIII, and XXIV. The 
figures for similar fracions from the complete Van Slyke analyses 
are added for comparison. These results will be discussed later. 



56 



Table XX. — "Jodidi numbers" determined on 100 cc. of hydrol- 
ysale of Fargo clay loam, together with a comparison of similar frac- 
tions taken from the Van Slyke analysis. 



Grfims nitrogen 



Per 



cent of total 
nitrogen 



Average 

data of 

Van Slyke 



TablrVin 
j Av. 



Total N \ . . . ^ . . 

Ammonia N 


I 
0.0484 
0.0160 


ir 

0.0478 
0.0160 


I 
' 33.06 


Residue from above acidi- 
fied with HCl and bases 
pptd. direct, "Basic N". 
N in filtrate from ''bases" 
Total N regained 


0.0033 
0.0315 
0.0508 


0.0055 
0.0277 
0.0492 


6.82 

65.08 

104.96 



u 

33.47 



11.51 

57.95 
102.93 



33.26 



9.16 

61.52 

103.94 



24.00 



9.58 
32.71 



Table XXL — "Jodidi numbers" determined on 200 cc. of hydrol- 
ysate of Fargo silt loam, together with a comparison of similar frac- 
tions taken from the Van Slyke analysis. 



Grams ^^\^^^' ''' 
nitrogen nit?o|en 


Average data 
of Van Slyke 
analysis. 
Table IX. 


Total N 1 0.1424 

Ammonia N | 0.0487 34.20 

Residue from alcove acidified 
with HCl and bases pptd. di- 
rect, "Basic N" 0193 13 55 


'26 56 

12 11 


N in filtrate from "bases" | 0.0777 54.56 

Total N regained | 0.1457 102.31 


35.14 



Table XXII. — "Jodidi numbers" determined on 100 cc. nf hydrol- 
ysate of Hempstead silt loam, together zuith a comparison of sim.ilar 
fractions taken from the J^an Slyke analysis. 





i 

1 Per cent of total 
Grams nitrogen | nitrogen, 


Average 
data of 

Van Sl.vke 
ana ysis 

Table XI 


Total N ; 

Ammonia N 

Residue from above acidi- 
fied with HCl and bases 
pptd. direct, "Basic N". 

N in filtrate from "bases" 

Total N regained 


I 

0.0487 
0.0207 

0.0068 
0.0243 
0.0518 


n 

0.0513 
0.0197 

0.0067 
0.0255 
0.0519 


I 

"42". 51 

13.96 

49.90 

106.36 


il 

'38.40 

13.06 

49.71 

101.17 


Av. 

' '40.45 

13.51 

49.81 

103.77 


' '29.44 
11.39 

28.10 



57 



Table XXIII. — "Jodidi numbers" determined on 200 cc. of hydrol- 
ysate of prairie-covered loess, together with a comparison of similar 
fractions taken from the Van Slyke analysis. 





Grams Per cent of 
nitrogen total nitrogen 


Average data 

of Van Slyke 

analysis. 

Table XII. 


Total N 


0.0601 
0.0244 

0.0036^ 
0.0321 


' 40.60 

5.99 

53.41 

100.00= 




Ammonia N 


30.53 


Residue from above acidified 
with HCl and bases pptd. di- 
rect, "Basic N" 


12.68 


N in filtrate from "bases" 

Total N regained 


28.80 







1 By difference. ^ Calculated. 

Table XXIV. — "Jodidi numbers" determined on 200 cc. of hydrol- 
ysate of forest-covered loess, together with a comparison of similar 
fractions taken from the Van Slyke analysis. 



Grams 
nitrogen 



Per cent 
of total 
nitrogen 



Average data 
1 of Van Slyke 
I analysis. 
1 Table XIII. 



Total N I 

Ammonia N I 

Residue from above acidified| 
with HCl and bases pptd. di-1 

rect, "Basic N" | 

N in filtrate from "bases" I 

Total N regained 



0.0340 
0.0130 



0.0059 
0.0175 
0.0364 




28.69 



13.98 
27.21 



17. Summary Tables. Certain of the preceding analyses have 
been summarized in Tables XXV, XXVI. In Table XXV are 
shown the amounts and percentages of soil dissolved by the acid 
during hydrolysis as well as the ainount of nitrogen and percentage 
of the total nitrogen dissolved. Table XXVI summarizes average 
nitrogen distribiition of Tables V, VI, VII, VIII, IX, X, XI, 
XII, XIII, XIV, XVI, XVII, XVIII, and XIX. 

Table XXV. — Percentages of soil and of soil nitrogen dissolved 
by hydrolyzing the different soil types. 





0) 


Grams 


Grams 


Per cent 


Grams 


Grams 


Percent 




a 




soil 


soil 


nitrogen 


nitrogen 


of dis- 


Soil type 


a 


taken 


dis- 


dis- 


in 


dis- 


solved 




to 


(dry basis) 


solved 


solved 


soil 


solved! 


nitrofjen 


Fargo clay loam 


I 

TT 


240.2 
240.2 


43.2 

43.2 


17.99 


0.6005 


0.4252 


70.79 


Fargo silt loam 


I 


222.8 


54.8 


24.60 


1.8336 


1.4237 


77.65 


Carrington silt loam . . 


I 


235.5 


36.5 


16.61 


0.8738 


0.6191 


70.91 


Hempstead silt loain . 


I 


242.3 


3.2.3 


13.33 


0.6201 


0.4395 


70.87 




TT 


242.3 


2,2,.^ 


13.74 




0.4508 


72,69 


Prairie-covered loess . 


T 


230.3 


42.3 


18.37 


0.6933 


0.5260 


75.91 




TT 


230.3 


45.3 


19.64 




0.4991 


72.02 


Forest-covered loess . 


T 


294.4 


29.4 


9.98 


0.3768 


0.2735 


72.19 




IT 


294.4 


32.4 


11.11 




0.2511 


66.65 



iThe figures in this column were obtained by subtracting the "insoluble 
humin nitrogen" remaining in the residual soil from the nitrogen figures ob- 
tained by multiplving the original ^weight of soil taken (dry basis) by the 
nitrogen content of the soil. These figures may or may not agree with tne 
figures obtained in kjeldahling a portion of the solution, due to experimental 
errors, and perhaps to errors introduced in using a uniform factor (.^i.b) ror 
specific gravity. The figures in this column are free from any error ot tnis 
sort. 



58 



•5 



isi 



"5 



CJi 






'^ 



^ 



•SS.XJI 



Xja.iaAO.) 



<U I— I 



puajscliuajj 



11 eiduius 



•J eiduiiiv^ 

ureoi :(ns 

nojSiu.i.iiiJ 



•LUIJOI 



•ureoi 



•tjos 

^.■![..>UUI,, 



•;b3(I-ssbj.§ 

IJOBiq BIIO3.T1S0IB0 



•TOH A' I 

■pjfl'-i HOBN %t 

u[ ajquios ^isad 

p,)jaAuJ-umu.uiJUdS 



•TOH A'l 

ui aiquios ^iMu 
p a.iaAO..)-umu.§«mIg 

•iO«.i;xa XOH %\ 

ui eiqiiios juad 

pa.xaAoJ -lumiSBiidy 



pd.iaAOO-iuuuSiiiid^ 



■l!(>s<liis +jv!ad 
paiaioo immSBiiaf: 



•j^ad 

pa.iaAuo-Miiiu.Siuid^ 



:x 









03 I I 



H 



3X 



HX 

hX 



HX 

<u 



ro LO 0\ -rr 00 
u-i ro t— I IT) \£) 

O ^' lO 0\ CNJ 
ro <M CM ^ 



00 00 t^ ""' * 



■ 0\ 



^ CM a\ -* ^ o o 

On i-O lO '— I ^ lO O 

00 i< ^ u^ fvi cvi o 



t^ MD "x|- (M OS fvl OO 



CM ^ C-1 00- 
O * * <^ O 

ioo'ro t^ O t^ 
"0 t^ 0\ C^ VO 

vd CM 00 ^ 00 






^ ,— I O ^ -^ ^ '-' 
CM <— I On O '-' i-O C^j 

ro -Nt- i-i O t< rrj C\i 



On 0\ On VO CM ^ oa 

OO C^l SO 0\ 00 >-; '^ 

VO t~^ <^ 00 ro lO CM 

c^ CO O 



LO SO t^ O LO LO 00 

C^ CM -* O -^ lO Ol 

so lO -vt- <-o O CM c^i 






o 
;-^ 

i a, 












fa ^ S O 



03 

mm m < 



o o;t:; C 



"z 





c 


^ 


bX) 














03 







03 


^ 


C 


c 


•^ 


C 


rt 







r^ 








;z;^<:2;H 



-M ?1 <u s 
cD.rt it!— • 



nO 



59 

18. An attempt to isolate pure proteins from a soil. From 
all the positive evidence it would seem that a very considerable 
portion of the nitrogen in the soil exists in other forms than pro- 
tein. For example, Potter and Snyder (1916) found an average of 
20.46 per cent of the alkali soluble nitrogen of a soil to be non- 
protein in nature. Bacteria and fungus spores all contain chitin 
which on hydrolysis yields glucosamine which will give amino 
(-NHo) nitrogen of non-protein origin. 

The average C/N ratio that exists in this series of six mineral 
soils studied has been shown by Gortner (1916 a) to be 12.23. The 
average nitrogen content of the six soils is 0.355 per cent which 
would indicate 2.22 per cent protein (NX6.25) if all of the nitrogen 
existed in this form. However, the total organic matter in these 
soils averaged 7.48 per cent (carbonXl.724), showing that less 
than 30 per cent of the soil organic matter could be of protein na- 
ture. This is shown equally v/ell by a comparison of the C/N 
ratios. The average analyses of sixteen plant proteins as recorded 
by Mathews (1915) gives carbon 52.08 per cent and nitrogen 17.73 
per cent. The average C/N in these vegetable proteins is 2.94. 
The high C/N ratio in soils indicates that the organic matter does 
not consist essentially of protein material (cf. however, Gortner 
1917, when a ratio of 3.06 was found nidicating that the organic 
matter in this instance was essentially protein). 

In view^ of the desirability of demonstrating the presence or 
absence of proteins, in soils an attempt was made to isolate from 
a soil either alcohol soluble or salt soluble proteins. A 6 inch 
bulk sample of Fargo silt loam from Morristown, Rice County, 
containing 0.397 per cent of nitrog-en in the air dry soil, was used. 

a. Extraction with 70 per cent ethyl alcohol. The soil was 
first leached with 1 per cent hydrochloric acid to the absence of 
calcium and washed with distilled water until practically all chlo- 
rfdes were removed. For this purpose sixty-one 100 gram por- 
tions Avere taken. It required about 150 liters of acid to remove 
all traces of calcium. 

After leaching, the soil was allowed to air dry in the green- 
house and there remained 5700 grams air dry soil. Five hundred 
gram portions were placed in twelve 2.5 liter acid bottles and ex- 
tracted successively with 70 per cent ethyl alcohol. A fresh por- 
tion of alcohol was added to the first bottle of the series, shaken, 
allowed to stand over night, syphoned ofT, and placed in the bottle 
next ahead in the series. This was continued until six successive 
portions of fresh alcohol had been added. The alcohol was ab- 
sorbed by the dry soil to such an extent that a 500 cc. portion of 
fresh alcohol had to be added to the bottles towards the end of 
the series. Almost 15 liters of alcohol were used, but only a little 
over 10 liters were regained at the end, since so much was retained 
by the soil. 

The combined extracts were filtered until practically free of 
clay and then concentrated under diminished pressure at a tem- 
perature of 55° C. The extracts were in all cases straw colored. 



60 

A heavy yellow precipitate separated on concentration but dis- 
solved very largely in the fresh portions of the alcohol extract 
The greater portion of water Avas finally removed by evaporating 
four times with 93 per cent alcohol. More water, however, could 
have been removed by evaporating with absolute alcohol. The 
trace of clay, remaining after concentration, was filtered off on a 
diy filter paper and the solution diluted to 500 cc. with 93 per 
cent alcohol. The total nitrogen was determined on duplicate 25 
cc. portions of the solution by the Kjeldahl method. The results 
gave 0.1320 gram nitrogen in the total alcohol extract. 

Tests were made on portions of this alcohol extract for the 
presence of proteins or protein-Hke substances. Precipitates were 
obtained with phosphotungstic acid and lead acetate. A heavy 
yellow precipitate formed on dilution with water. This yellow 
precipitate was readily soluble in sodium hydroxide (suggests 
presence of acids or phenols), and in concentrated hydrochloric 
acid. The biuret test and Liebermann's reaction both gave nega- 
tive tests, and Millon's reaction gave an extremely faint test (mere 
trace) . 

One 25 cc. portion of the alcohol solution was diluted with 
water and extracted four successive times with chloroform, fol- 
lowed by a single extraction with ether. The chloroform and ether 
extracts were combined and evaporated to dryness in a platinum 
dish on the steam bath and later in a hot air oven at 102° C. and 
the residue weighed. The results indicated the presence of 0.3108 
gram of organic matter, making a total of 6.2160 grams in the en- 
tire alcohol extract. 

After the chloroform-ether extraction there remained a dark 
brown granular insoluble substance. This was filtered off on a 3 
cm. Buchner funnel and the filter and precipitate used for the 
determination of nitrogen. The results indicated the presence of 
0.0010 gram maximum protein nitrogen in 'this solution, leaving 
0.1310 gram of non-protein nitrogen in the same extract. 

The negative color tests and the small amount of nitrogen 
indicate that no protein is present in this soil that is soluble in 
alcohol. 

Another 25 cc. portion of the alcohol extract was evaporated 
to dryness on the water-bath in i platinum dish and subsequently 
dried'in an oven at 96° C. with a short final heating at 103° C. The 
organic matter amounted to 0.6126 gram, making a total content 
of organic matter in the alcohol solution 12.2520 grams. From the 
data given it .was found that onlj 1.08 per cent of the organic mat- 
ter in the 70 per cent alcohol ext -act of the soil was nitrogen. 

b. Extraction with absolute alcohol. A 100 gram portion -of 
the unleached soil was extracted with absolute alcohol for 60 
hours in a Soxhlet extraction apparatus. The extraction flask had 
a capacity of 500 cc. and all the joints of the apparatus were ground 
glass. Several pieces of broken porcelain were placed in^ the flask 
to prevent bumping, as the amount of dissolved organic matter 
increased. An alundum extraction thimble was used to hold the 



61 

soil. The alcohol syphoning- back became colorless several hours 
before the extraction was stopped. At the end of the extraction 
the solution in the flask was deep straw color. 

The total alcohol extract was evaporated to small volume and 
then the total nitrogen determined, which was found to be 0.0011 
gram. This was equivalent to 0.0660 gram of nitrogen in 6 kilo 
of soil before leaching with acid. It will be recalled that 0.1320 
gram nitrogen dissolved in the 70 per cent alcohol after first leach- 
ing with 1 per cent hydrochloric acid. 

This would indicate that many of the organic nitrogenous 
compounds are in combination with the liine or other bases present 
in the soil, and that this combination is broken up when leached 
with 1 per cent- hydrochloric acid. 

c. Extraction with 10 per cent sodium chloride. After com- 
pletion of the alcoholic extraction, the soil residue was dried in an 
air oven at about 65° C. and placed in a 20 liter bottle. To this 
was added 2000 cc. of 10 per cent sodium chloride solution for 
each kilo of dry soil. The bottle was shaken repeatedly and allowed 
to settle over night. The presence of an electrolyte caused the 
clay to settle rapidly, so that a clear solution could be withdrawn. 
After the soil had been in contact with the salt solution 20 hours, 
two 1(X) cc. portions were withdrawn and analyzed for nitrogen. 
1'he results showed that 0.1350 gram nitrogen was contained in 5 
liters of the supernatant liquid. After shaking and standing for 
three days duplicate determinations were again made on 100 cc. 
portions with the result that 0.1600 gram nitrogen M^as present in 
the corresponding solution. This inclicates that the longer the soil 
is in contact with the sodium chloride solution the greater the 
amount of organic nitrogen extracted. 

Two .500 cc. portions of the salt extract were used for precipi- 
tation with phosphotungstic acid. After the addition of 45 cc. of 
concentrated hydrochloric acid, the phosphotungstic acid was 
added. The gelatinous i^recipitates formed were filtered on an 
ordinary funnel and washed with the filtrate, and then used for 
the determination of nitrogen by the usual method. The duplicates 
averaged 0.0028 gram nitrogen, thus making the total nitrogen 
precipitated by phosphotungstic acid 0.0280 gram in the 5 liters of 
supernatant lic|uid. This shows that 17.50 per cent of the nitrogen 
extracted from the residual soil was precipitated by phosphotung- 
stic acid. This represented the maximum protein nitrogen in the 
10 per cent sodium chloride solution. 



62 

III. DISCUSSION. 

A. Changes in nitrogen distribution in a protein when hydro- 
lyzed in the presence of a mineral soil. From a study of Table 
III it is seen that the histidine nitrogen formed when fibrin is 
hydrolyzed alone is 4.36 per cent and when hydrolyzed in the pres- 
ence of cellulose it is 4.86 per cent of the total nitrogen. By re- 
ferring to Table II where hbrin is hydrolyzed in the presence of 
ignited subsoil we see the histidine nitrogen is entirely lacking and 
that the nitrogen precipitated by calcium hydroxide amounts to 
4.81 per cent. This corresponds very closely to the amount of 
histidine found in the other two cases. In other p(Mnts the three 
analyses agree within experimental error. 

It appeared possible that the histidine nitrogen might have 
been converted into the nitrogen fraction precipitated by calcium 
hydroxide. It is a well known fact that histidine can be precipi- 
tated by silver nitrate in slightly alkaline solutions. Since there 
are a large number of mineral constituents in the soil it may be 
possible that the histidine could be precipitated by some of these 
and thus be found with the calcium hydroxide precipitate. 

With this idea in mind an analysis of histidine was made in 
the presence of an ignited subsoil, only three fractions being de- 
termined. One 0.5000 gram sample of histidine di-hydrochloride* 
and 50 grams ignited subsoil were boiled in the presence of 100 
cc. of hydrochloric acid (sp. gr. 1.18) for 48 hours. The solution 
was diluted to 500 cc. in a graduated flask and two 200 cc. portions 
syphoned off and analyzed. The solution was deep straw color. 
The determinations for ammonia nitrogen gave negative results. 
The precipitate formed by calcium hydroxide was washed by de- 
cantation until free of dissolved nitrogen compounds, and the total 
nitrogen determined. This precipitate was bulky due to the pres- 
ence of large amounts of ferric and aluminum hydroxides. The 
average humin nitrogen in the two samples was only 0.0006 gram. 

The filtrate from the calcium hydroxide precipitate was con- 
centrated to a small volume and the entire solution used for the 
nitrogen determination. .Sample I, contained 0.0371 gram in the 
filtrate, and Sample II, 0.0365 gram, making an average of 0.0368 
gram. 

The residual soil was practically colorless, and a determina- 
tion indicated that it was nitrogen free. The volume occupied by 
the soil residue was 17.3 cc. By calculation it was fmmd that the 
total nitrogen regained in the original solution was 0.0903 gram, 
theoreticaf 0.0921 gram, or a recovery of 98.01 per cent. 

Thus practically all of the histidine was recovered in the fil- 
trate from the calcium hydroxide precipitate, indicating that the 
hypothesis was incorrect. 

*The histidine^ di-hydrochloride was prepared from dried blood as out- 
lined by Abderhaldeii (1910). Total nitrogen found 18.42 per cent; calculated 
18.42 per cent. 



63 

It is possible that" the specilic character of the histidine nitro- 
gen is destroyed by certain oxidizing agents present during the 
hydrolysis, with the result that histidine nitrogen is not precipi- 
tated in the basic fraclion. A study is being conducted at the pres- 
ent time on the hydrolysis of pure protein in the presence of certain 
inorganic oxidizing agents. It is hoped that some light will be 
thrown upon the disappearance of histidine as well as the formation 
of "humin" and of the "nitrogen precipated by calcium hydroxide." 

It is of interest to note that the average total nitrogen in the 
two samples in Table II is 0.4584 gram (of. Gortner 1916 c, where 
total nitrogen on four determinations of three grams averaged 
0.4551), showing that all of the nitrogen present is accounted for 
in the method of analyses used. 

Table IV shows the difference between the duplicate deter- 
minations of the analysis of fibrin alone and in the presence of car- 
bohydrate and of subsoil, and the differences apparently due to the 
addition of 100 grams ignited subsoil to the 3 grams of fibrin. Van 
Slyke's (1911) "maximum" and "average" differences to be ex- 
pected between duplicate determinations are also given in the table 
for reference. 

From a study of Table IV it Avill be seen that the difference 
between the analyses of fibrin hydrolyzed alone and in the presence 
of ignited subsoil are, in the case of most of the fractions, within 
the maximum allowed by Van Slyke for experimental error. The 
only differences which are certainly greater than experimental 
error are those of humin nitrogen, histidine nitrogen, and amino 
nitrogen in the filtrate from the bases. It is observed that prac- 
tically the same error occurred with the amino nitrogen in the fil- 
trate from the bases when hydrolysis was carried out in the pres- 
ence of carbohydrate. 

From this analysis one can only draw the conclusion that even 
if the organic matter of the soil consisted entirely of pure protein, 
one would not obtain the same nitrogen distribution by the Van 
Slyke analysis in the presence of soil that one would obtain in the 
absence of the soil, or in other wo^rds, the presence of ignitied min- 
eral subsoil interferes with the Van Slyke analysis in much the 
same manner as carbohvdrates (Gortner 1916 c. and Hart and Sure 
1916). 

B. The humin nitrogen, its origin and significance. In such 
a discussion one must first consider the source of humin nitrogen 
in pure proteins. 

Osborne and Jones (1910) suggest that perhaps tryptophane 
and histidine are responsible for the humin formation, basing their 
postulation on the fact that zein, which contains no tryptophane 
and but little histidine. gives only small amounts of humin on 
hydrolysis. 

Gortner and Blish (1915) hydrolyzed zein in the presence of 
both tryptophane and of histidine and found that a large part of 
the tryptophane was converted into humin nitrogen, whereas none 



64 

of the histidine was converted into huniin but was all recoverable 
in the bases. I have shown that histidine is practically all recov- 
ered in the filtrate from the humin when it is hydrolyzed in the 
presence of an ignited mineral subsoil. Histidine, therefore, can 
be eliminated as a factor in the formation of humin nitrogen in the 
soil. Gortner and Blish conclude that — 

in all probability the humin nitrogen oi protein liydrolysis has its origin in 
the tryptophane nucleus. 

Gortner (1916 c) has shown that the humin nitrogen is in- 
creased by the addition of carbohydrate material to protein, and 
suggests that this increase may be due to both physical and chem- 
ical causes.* He presents evidence to show that the action of car- 
bohydrate is probably due to the furfural produced from the car- 
bohydrate and shows that increasing quantities of furfural cause 
the humin nitrogen to steadily increase, and the work of Gortner 
and Holm ( 1917) shows that the presence of formaldehyde during 
hydrolysis causes a gradual increase in the amount of humin nitro- 
gen up to a maximum very much larger than the amount of normal 
humin nitrogen, and then a decrease with increased amounts of 
aldehyde. 

Shmook (1914) states that during the hydrolysis of his soils 
there separated on the walls of the condenser a substance violet blue 
in color, and that this appears during the hydrolysis of pure pro- 
tein and is recognized as Liebermann's reaction for protein sub- 
stances. The above conclusion in regard to the hydrolysis of a 
pure protein is incorrect, since no color appears on the neck of the 
flask or condenser in such an analysis. When furfural is heated 
alone with hydrochloric acid a characteristic colored substance is 
deposited on the condenser. It has been shown by Gortner (1916 
c) that at the same time a polymerization (?) of furfural to humin 
takes place very rapidly. He found that when 1.165 grams of 
furfural were heated with 100 cc. of hydrochloric acid (sp. gr. 
1.115) for 18 hours that 76.40 per cent of the original furfural was 
converted into insoluble "humin." Our mineral soils on hydrolysis 
gave a deposit on the condenser similar to that described by 
Shmook. The reaction indicates the presence of furfural, which 
is in turn formed from the carbohydrates in the soil. This must 
be considered to be a distinctive furfural reaction. 

The humin nitrogen actually present in the soil may easily be 
a very small part of the nitrogen found. It is evident that there 
must be many nitrogenous organic compounds present in the soil 
which have no relation to protein material, such as purine, pyrimi- 
dine bases, nitrogenous lipins, and nitrogenous pigments besides 
a number of other non-protein substances. It is certain that the 
humin nitrogen will be greatly changed by the presence of many 
of these compounds. The calcium hydroxide here drags down all 
the organic nitrogenous compounds which are soluble in dilute 
acids, but insoluble in hot water and dilute calcium hydroxide, to- 

*Practically the same increase in humin nitrogen occured when fibrin 
was hydrolyzed in the presence of a mineral subsoil. The humin in this 
case was not due to the presence of carbohydrate since the soil had lost all of 
its organic matter by ignition. 



65 

gether with the calcium salts of nitrogenous organic acids, the 
calcium salts of the purine and pyrimidine bases in addition to 
the humin formed from the protein material, and other organic 
compounds that are adsorbed, absorbed, occluded, or combined with 
the iron and aluminum hydroxides present. 

From Table XXVI we find that from 4.84 to 9.21 per cent of 
the total nitrogen is precipitated by calcium hydroxide. It can 
readily be seen that this does not represent true humin nitrogen, 
since the calcium hydroxide does not contain any black colored sub- 
stances formed by hydrolysis. The solution from which it is pre- 
cipitated is colored only by ferric compounds, therefore, the or- 
ganic material in this precipitate must consist of colorless organic 
compounds adsorbed by or combined with the lime. This por- 
tion of the nitrogen consists almost certainly of non-protein mate- 
rial. In all pure proteins the nitrogen retained in the calcium hy- 
droxide precipitate is supposed to consist entirely of deeply colored 
compounds. This study of the distribution of organic nitrogen 
in the soil has led to a new fraction, not previously reported. Cer- 
tain of the analyses were carried out before the importance 
of this fraction was realized, but in most of the analyses I have 
reported this fraction as "nitrogen precipitated by calcium hy- 
droxide," because of its unknown nature. Further investigations of 
this fraction are highly desirable. 

Another point of interest is observed in the humin nitrogen of 
the sphagnum-covered peat hydrolyzed in the presence of metallic 
tin. There is a decided decrease in this fraction as compared with 
the peat hydrolyzed alone. As noted earlier, Samuely (1902), sug- 
gested that humin formation might be due to an oxidation process 
and certain of the earlier workers (of. Hlasiwetz and Habermann 
1871 and 1873) hydrolyzed protein in the presence of stannous chlo- 
ride in order to obtain a colorless solution instead of one deeply 
colored by the presence of humin. 

I, therefore, hydrolyzed some gliadin in the presence of tin and 
found that while the solution remained colorless, nevertheless small 
balls of black material were formed. The humin nitrogen (insolu- 
ble in acid -f- that pptd. by Ca(OH)2) was 1.17 per cent of the total 
nitrogen, while gliadin hydrolyzed alone gave a dark colored hy- 
drolysate and a humin nitrogen content of only 0.67 per cent. 

Recently Spriestersbach (private communication) has hydro- 
lyzed fibrin (from a different sample than mine) alone and in the 
presence of stannous chloride and finds in the fibrin hydrolyzed 
alone 1.67 per cent of total humin nitrogen, of which 1.06 per cent 
is "acid insoluble" (of. Gortner 1916 c) and 0.61 per cent precipi- 
tated by calcium hydroxide. In the sample of fibrin hydrolyzed 
in the presence of tin he finds 0.91 per cent of total humin nitrogen, 
of which 0.25 per cent is "acid insoluble" and 0.66 per cent pre- 
cipitated by calcium hydroxide. The nature of his hydrolysate 
agreed in all respects with mine, i.e., was colorless or faint straw 
color, with tiny black balls of humin floating on the surface or at- 



66 

tached to the sides of the flask in which the hydrolysis was carried 
out. 

These experiments suggest interesting- possibilities, but dis- 
cussion must be deferred until we know more of the exact reac- 
tions taking place. 

The true humin nitrogen remains in the residual soil after 
hydrolysis. The amount of nitrogen in this fraction varies from 
22.93 per cent to 28.27 per cent of the total nitrogen for the mineral 
soils studied. This represents more nearly the true humin nitrogen, 
in that the black coloring matter formed by acid hydrolysis remains 
in this portion, but in addition we should also find here all organic 
nitrogenous compounds insoluble in fairly strong solution of hy- 
drochloric acid, all of the nitrogen adsorbed by the carbohydrate 
humins, etc. Potter and Snyder (1915 a) express surprise at the 
large proportion of nitrogen in this fraction but when one considers 
the heterogeneous nature of the soil organic matter it is perhaps 
more surprising to find that over 60 per cent of the nitrogenous 
compounds are soluble in strong hydrochloric acid. Further study 
is necessary before the full significance and origin of this humin 
nitrogen can be thoroughly understood. 

C. The effect of the quantity of acid used for the hydrolysis 
on the amount of nitrogen dissolved and the nitrogen distribution 
in soils. Throughout this investigation acid at least as strong as 
constant boiling hydrochloric acid was used for the hydrolysis 
since that is the strength recommended for the analysis of pure 
proteins. 

In the case of two soils, however, one of the duplicates was 
hydrolyzed in the presence of 1000 cc. concentrated acid to 250 
grams of soil, the other being hydrolyzed in the presence of 500 
cc. of constant boiling acid to 250 grams of soil, in order to see if- 
any noticeable differences would be observed on the resulting analy- 
ses. The two soils thus hydrolyzed were the prairie-covered loess 
and forest-covered loess. 

The res^ults show little difference between the duplicates. 
Table XXV shows that the larger volume of the stronger acid dis- 
solved a greater per cent of the soil, due to the fact that more of 
the mineral constituents were soluble in acid of this concentration. 
At the same tim.e, however, the amount of nitrogen extracted was 
less. 

D. The percentage of soil nitrogen extracted by acid hydro- 
lysis. Shorey (1905) working with a single Hawaiian soil ex- 
tracted 84.68 'per cent of the total soil nitrogen by acid hydrolysis. 
Jodidi (1911) working wnth eleven Iowa soils found from his 
studies a minimum of 68.90 per cent, a maximum of 83.94 per cent, 
and an average of 75.77 per cent; Lathrop and Brown (1911) iii 
five Pennsylvania soils found a minimum of 70.60 per cent, a maxi- 
mum of 73.71 per cent, and an average of 71.78 per cent; Shmook 
(1914), working with four Russian soils, found a minimum of 
60.60 per cent in the Laterite soil, a maximum of 87.67 per cent in 
the Podzol soil, and an average of 68.33 per cent; Kelley (1914), 



67 . 

working with nine soils of the Laterite class common to the Ha- 
waiian Islands, found a minimum of 67.51 per cent, a maximum 
of 91.80 per cent, and an average of 82.17 per cent; and Potter and 
Snyder (1915 a) in seven Iowa soils, found a minimum of 68.68 
per cent, a maximum of 76.47 per cent, and an average of 74.41 
per cent. 

The grand average of all of these thirty-seven soils from wide- 
ly different origin gives 75.91 per cent of the soil nitrogen in solu- 
tion in the hydrochloric acid extract. In my studies I found a 
minimum of 66.63 per cent, a maximum of 77.65 per cent, and an 
average of 72.19 per cent extracted by the acid. 

These results indicate that the nitrogen of practically all soils, 
in so far as investigated, dissolves to about the same extent during 
the acid hydrolysis. 

E. "Jodidi numbers." A study of Tables XX, XXI, XXII, 
XXIII, and XXIV shows that the' nitrogen distribution by this 
method does not give accurate results when compared with similar 
fractions of the Van Slyke analyses. The ammonia nitrogen and 
nitrogen in the filtrate from "bases" are all much too high, while 
"basic N" corresponds fairly well with the true basic nitrogen. 
If one desires accurate data in regard to the distribution of the 
organic nitrogen in the soil he should not use "Jodidi numbers." 

F. Attempts to extract proteins from the soil. The attempt 
to isolate alcohol or salt soluble proteins from the soil was not suc- 
cessful. The maximum protein nitrogen in the 70 per cent alcohol 
extract from 6 kilo of soil amounted to^ only 0.0010 gram, while 
the maximum protein nitrogen extracted by the 10 per cent sodium 
chloride amounted to 0.0280 gram or 17.50 per cent of the total 
nitrogen in solution. A larger amount of organic nitrogen was 
extracted by alcohol when the soil was first leached with 1 per cent 
hydrochloric acid. 

The amounts of possible protein were so small that it seems 
safe to conclude that no appreciable quantities of alcohol soluble 
or salt soluble proteins are found in the soil. 

G. A consideration of the nitrogen distribution in different 
extracts froiTi the sphagnum-covered peat. Nitrogen distribution 
was determined on extracts of a sphagnum-covered peat soluble 
in (a) 1 per cent hydrochloric acid, (b) 4 per cent sodium hydroxide 
and not precipitated by acidification, and (c) 4 per cent sodiuni 
hydroxide and precipitated by acidification Avith hydrochloric acid. 
Of the three extracts only the second approximates the distribution 
of nitrogen in a pure protein. The figures for the ammonia nitro- 
gen are abnormally high in the hydrochloric acid extract. 

The humin nitrogen is high in all the extracts, but is excessive 
in fraction (c). It is clear that carbohydrates from the soil must 
be present in all three fractions used, and must have some share 
in bringing the humin nitrogen up to such high figures. The 
nucleic acids (Shorey 1911a, 1912) would be found in the hydro- 
chloric acid precipitate from the sodium hydroxide solution, and 



68 



the purine and pyrimidine compounds of these nucleic acids, as 
well as the lecithins (Aso 1904, Stoklasa 1911) and nitrogenous 
lipins and nitrogenous acids would be precipitated with the true 
humin by the calcium hydroxide. 

The basic nitrogen figures are not widely divergent although 
there may be some significant differences. The differences be- 
tween the nitrogen in the filtrate from the bases is perhaps the most 
significant of all. An amino nitrogen of only 17.11 per cent in the 
filtrate from the bases such as is found in the hydrochloric acid 
extract is far lower than has ever been obtained in an analysis 
of a pure protein and indicates that the nitrogen of this extract is 
essentially non-protein. 

Unfortunately it was impossible to complete the corresponding- 
analyses on the calcareous black grass-peat, but the fractions ob- 
tained would indicate a distribution similar to that of the sphagnum- 
covered peat. 

H. General conclusions in regard to the distribution of soil 
nitrogen in different soil types. From a study of Table XXVI vve 
observe a great similarity between the different fractions. This is 
practically the same deduction made by Potter and Snyder (1915 a) 
in regard to their study made on a single soil type under different 
fertilizer treatment. 

I find that the nitrogen distribution in a soil is very uniform 
whether in the same soil type under different fertilizer treatment, 
or in different soil types. This is to be expected, for if one were 
to take fifty Van Slyke analyses of protein at random and compare 
the average analyses with that of another fifty analyses, one should 
expect to find results agreeing closely with each other. This 
expectation should also hold true for the hydrolysate of soils, since 
in each soil are to be found many of the nitrogenous compounds 
contained in the plant and animal products that find their way 
to the soil together with their decomposition products. Since 
there is such a great variety of different nitrogenous substances 
in the soily it stands to reason that the nitrogen distribution in 
soils is an average distribution, and as such should not be ex- 
pected to vary widely from soil to soil. 

It has been shown in the earlier part of this discussion that 
when fibrin was hydrolyzed in the presence of ignited subsoil, no 
histidine fraction was obtained. 

For reasons which were stated previously, I have not tabu- 
lated the nitrogen distribution under the different headings used 
for the analysis of pure proteins. However, in view of the results 
obtained on the fibrin hydrolyzed in the presence of ignited sub- 
soil, it is perhaps worth while to consider what values the histidine 
fraction would have had. The Fargo clay loam, Fargo silt loam, and 
Sample I of the Carrington silt loam, gave results indicating that 
this fraction was absent, while Sample II of Carrington silt loam, 
gave 2.97 per cent, Hempstead silt loam 0.78 per cent, prairie-cov- 
ered loess 1.21 per cent, and forest-covered loess 1.25 per cent of 
histidine nitrogen. 



69 

Potter and Snyder (1915 a) find this fraction to be present in 
all of their eight soils, with a minimum of 1.99 per cent and a maxi- 
mum of 6.30 per cent. They do not give sufficient analytical data 
to permit a recalculation of their figures in order to ascertain if 
any errors in calculation were made. However, it is possible that 
their finding is due to the fact that all of their mineral soils repre- 
sented a single soil type. It is possible that this form of nitrogen 
may have been especially abundant in their particular soil. It is 
of particular interest to note that their soil was from the Wisconsin 
drift area, as was my sample of Carrington silt loam from Morris- 
town, which gave my maximum amount of nitrogen in this frac- 
tion. The sample of Carrington silt loam from Nerstrand was sit- 
uated on the Kansan drift and gave no "histidine" nitrogen. 



70 



IV. SUMMARY. 

This paper deals with a study of the nitrogen distribution in 
different soil types, by applying Van Slyke's method. Tables have 
been presented showing such distribution lor the following mate- 
rials : 

a. Fibrin hydrolyzed in the presence of an ignited mineral 

subsoil (together with data of fibrin hydrolyzed alone 
and in the presence of carbohydrates). 

b. A calcareous black grass-peat. 

c. An acid, sphagnum-covered peat, hydrolyzed alone, in the 

presence of a mineral subsoil, and in the presence of 
stannous chloride. 

d. An acid "muck" soil. 

e. Seven samples of mineral surface soil representing the fol- 

lowing soil types : Fargo clay loam. Fargo silt loam, 
Carrington silt loam (two samples from different glacial 
drifts), Hempstead silt loam, prairie-covered loess, and 
forest-covered loess. 

f . Extracts of a spliagnum-covered peat and of a calcareous 
black grass-peat soluble in (a) 1 per cent hydrochloric 
acid, (b) 4 per cent sodium hydroxide but precipitated 
by acid, and (c) 4 per cent sodium hydroxide and not 
precipitated by hydrochloric acid. 

The following conclusions are evident : 

1. The figures for the ammonia nitrogen in a protein analysis 
are not appreciably changed when the hydrolysis is carried out in 
the presence of an ignited mineral soil equal to twenty times the 
weight of the protein material. 

2. The "humin" nitrogen is greatly increased by the addition 
of ignited mineral soil. It was shown that histidine nitrogen cannot 
account for this increase, neither is it due to ^ the presence of car- 
bohydrates, since the soil lost all its organic matter on ignition. 

3. Attention has been called to the fact that the analysis of 
a pure protein in the presence of even an ignited mineral soil does 
not give reliable results for the different fractions, and that such 
determinations are of value only when used for purposes of com- 
parison. Such data should not be compared with analyses ot pure 
proteins. 

4. Since practically all mineral soils give furfural on treat- 
ment with acid it is very likely that a very considerable amount 
of the total humin nitrogen found is due to the presence of carbohy- 
drates in the soil, which give rise to furfural during hydrolysis. 
This may combine with certain of the nitrogenous compounds and 
cause an increase in the "humin" nitrogen, as well as adsorb or 
occlude nitrogenous compounds in the "humin" formed from fur- 
fural by polymerization. 



71 

5. This investigation of the distribution of organic nitrogen 
in the soil indicates a new fraction, the nature of which has not 
been previously recognized. This is the fraction of nitrogen re- 
moved from a colorless solution by calcium, iron, and aluminum 
hydroxides on the addition of calcium hydroxide. The nitrogen 
retained in this fraction must consist almost entirely of non-pro- 
tein material, since the organic substances in this precipitate have 
been shown to be colorless organic compounds adsorbed by or 
combined with the metallic hydroxides. This fraction has been 
reported as nitrogen precipitated by calcium hydroxide. 

6. The true humin nitrogen remains in the residual soil after 
hydrolysis, but in addition non-humin nitrogenous conipounds 
must also be retained in this fraction. 

7. The strength and volume of the hydrochloric acid used in 
hydrolysis has little effect on the nitrogen distribution of the hy- 
drolysate provided acid as strong as constant boiling acid is used, 
in the proportion of at least two parts of acid to one of soil. 

8. Results gained from a study of different soils indicate that 
the organic nitrogen dissolves, during hydrolysis, to almost the 
same extent regardless of the origin and nature of the soil. 

9. Some very interesting figures are found in the comparison 
of the different extracts from sphagnum-covered peat (Table 
XXVI). The portion soluble in sodium hydroxide and not pre- 
cipitated by hydrochloric acid gives a nitrogen distribution ap- 
proximating very closely that of a normal plant protein. The nitro- 
gen dissolving in the preliminary hydrochloric acid leaching shows 
a nitrogen distribution which is certainly not due exclusively to 
protein materials, e. g., an ammonia nitrogen percentage of 65.40 
and amino-nitrogen-in-filtrate-from-bases of 17.11 per cent. 

10. When an attempt was made to isolate alcohol soluble 
and salt soluble proteins from the soil the amounts obtained were 
so small that it seems safe to conclude that no appreciable quan- 
tities of these types of proteins are present. 

11. The most significant fact brought out by this study is 
that the organic nitrogen distribution in different soil types is very 
uniform. This is to be expected since it has been pointed out that 
the nitrogen distribution in soils is an average distribution of all 
the plant and animal nitrogenous products that find their way to 
the soil. 



72 



V. LITERATURE CITED 

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72> 

Detmer, W. 

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DOJARENKO, A. 

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Effront, Jean. 

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BIOGRAPHICAL. 

Clarence Austin Morrow was born near Morrow, Warren 
County, Ohio. He graduated from the Hillsboro, Ohio, high school 
in June, 1901. He entered the Ohio Wesleyan University in the 
Jail of 1902, and received the degree of Bachelor of Science in June, 
1906. During 1906-08 he held the position of Assistant in Chem- 
istry at Oberlin College. He received the degree of Master of Arts 
from the same institution in June, 1909. In 1909-10 he was acting 
head of the Departments of Chemistry and Physics at Doane Col- 
lege. Having been appointed the John Harrison Scholar in Chem- 
istry at the University of Pennsylvania he entered that institution 
in the fall of 1910 and continued his graduate work. In the spring", 
of 1911 he was appointed John Harrison Fellow in Chemistry, but 
later resigned this to take the position of Professor of Chemistry 
at Nebraska Wesleyan University, which position he has held to 
date (January, 1917). During 1914-15 he was granted leave of 
absence for study in the Division of Soils, University of Minnesota, 
where he held the position of Assistant in Soil Chemistry. Here 
he studied for the degree of Doctor of Philosophy. 

Major subject, soil chemistry. 

Minor subject, organic chemistry. 

Member of the American Chemical Society. 

Member of the American Association for the Advancement of 
Science. 



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